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Midazolam is a benzodiazepine with sedative, muscle relaxant, anxiolytic, and anticonvulsant effects. Twelve ball pythons (Python regius) were used in a parallel study evaluating the pharmacokinetics of 1 mg/kg midazolam following a single intracardiac (IC) or intramuscular (IM) administration. Blood was collected from a central venous catheter placed 7 days prior, or by cardiocentesis, at 15 time points starting just prior to and up to 72 hr after drug administration. Plasma concentrations of midazolam and 1-hydroxymidazolam were determined by the use of high-performance liquid chromatography tandem-mass spectrometry and pharmacokinetic parameters were estimated using noncompartmental analysis. The mean ± SD terminal half-lives of IC and IM midazolam were 12.04 ± 3.25 hr and 16.54 ± 7.10 hr, respectively. The area under the concentration-time curve extrapolated to infinity, clearance, and apparent volume of distribution in steady-state of IC midazolam were 19,112.3 ± 3,095.9 ng*hr/ml, 0.053 ± 0.008 L hr -1  kg -1 , and 0.865 ± 0.289 L/kg, respectively. The bioavailability of IM midazolam was estimated at 89%. Maximum plasma concentrations following an IM administration were reached 2.33 ± 0.98 hr and 24.00 ± 14.12 hr postinjection for midazolam and 1-hydroxymidazolam, respectively, and 22.33 ± 20.26 hr postinjection for 1-hydroxymidazolam following IC administration., (© 2019 John Wiley & Sons Ltd.)