Background and Objective: Trofinetide is the first drug to be approved for the treatment of Rett syndrome, a neurodevelopmental disorder. The purpose of the study is to fully characterize the metabolic and excretion profiles of trofinetide in humans. Methods: This Phase 1, open-label, single-dose trial conducted in healthy male adults was designed to characterize the pharmacokinetics of trofinetide (absorption, metabolism, and excretion), mass balance of [14C]-trofinetide, and safety profile of trofinetide following administration of an oral 12-g dose administered as a mixture of trofinetide and [14C]-trofinetide. Blood, urine, and fecal samples were collected at prespecified timepoints. The pharmacokinetics of trofinetide were assessed in blood and urine samples using high-performance liquid chromatography (HPLC) with tandem mass spectrometric detection. Bioanalysis of radioactivity was conducted in blood, plasma, urine, and fecal samples using liquid scintillation counting. Metabolite profiling was conducted in blood, plasma, urine, and fecal samples using HPLC with liquid scintillation counting of chromatographic fractions. Safety and tolerability, including treatment-emergent adverse events (TEAEs), were assessed. Results: Blood concentration-time profiles of trofinetide and total radioactivity were almost superimposable up to ~12 h after dosing. Urine concentration-time profiles of trofinetide and total radioactivity were similar. Trofinetide was rapidly absorbed into the circulation with an initial rapid decline (half-life [t½] alpha ~2.6 h), followed by a relatively slow terminal elimination phase (t½ beta ~20 h). The blood-to-plasma total radioactivity ratios were 0.529–0.592, indicating a lack of affinity for the cellular portion of blood. Renal excretion accounted for 83.8% of the administered radiochemical dose; 15.1% was recovered in feces. Urine and fecal recovery of radioactivity accounted for 99% of the administered dose at 168 h after dosing. Parent [14C]-trofinetide was the major radiolabeled entity in blood and plasma (88.4% and 93.1% in area under the concentration–time curves from 0 to 12 h [AUC0–12] in pooled blood and plasma samples, respectively) and the major entity excreted in urine (91.5% in 0–48-h pooled urine samples) and in feces (52.7% in 0–192-h pooled fecal samples). Only small levels of metabolites were present. In blood and plasma, only two minor metabolites were identified (each metabolite ≤ 2.24% of the AUC0–12 pool). These two metabolites were also observed in urine and fecal samples (≤ 2.41% of dose). In feces, one additional metabolite (0.84% of dose) was identified. Two mild TEAEs were reported in two participants and were not considered related to trofinetide. There were no clinically meaningful changes in individual laboratory parameters, vital signs, physical findings, or electrocardiogram results. Conclusions: Metabolic and excretion profiles confirm that trofinetide undergoes minimal hepatic or intestinal metabolism and is primarily excreted unchanged in the urine. Trofinetide containing radiolabeled [14C]-trofinetide was well tolerated. Plain Language Summary: Trofinetide is the first approved treatment for Rett syndrome, a rare genetic condition that affects brain development. Study aims were to look at how a single oral dose of trofinetide is absorbed into the bloodstream, to see whether trofinetide's chemical structure is changed once in the body, and to see how trofinetide and any metabolites (chemically altered trofinetide) are removed from the body. Safety and tolerability of trofinetide were also assessed. Eight healthy adult men took a single oral 12-g dose administered as a mixture of 14C-radiolabeled and nonlabeled trofinetide. Researchers collected blood, urine, and stool samples at regular intervals for up to 10 days postdose to measure levels of trofinetide and its metabolites. Trofinetide was rapidly absorbed (time to maximum concentration was 2 h postdose) and was primarily present in the blood as the unaltered compound. Concentrations decreased rapidly during the first 24 h postdose and more slowly thereafter. Most of the dose was recovered in urine with a lower amount in stool samples (83.8% and 15.1% of the radiochemical dose, respectively). Total recovery in urine and stool samples was 99%, primarily as the chemically unaltered compound. Only low levels of three trofinetide metabolites were detected. Two metabolites were found in blood, urine, and stool samples, while one metabolite was found in stool samples only. Two mild treatment-emergent adverse events, considered to be unrelated to trofinetide, were reported. In summary, trofinetide is rapidly absorbed, minimally metabolized, and mainly removed from the body in the urine as the unchanged drug. [ABSTRACT FROM AUTHOR]