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Background: Electronic cigarettes (e-cigs) are a form of tobacco product that has become increasingly popular over the past decade. Despite the known health consequences of tobacco product exposure during pregnancy, a substantial number of daily smokers will continue to smoke during pregnancy. Our current knowledge on the effects of e-cig aerosol exposure during pregnancy is limited to a small number of animal studies, which have identified several e-cig aerosol-induced disruptions to the physiology of normal development. Methods: To further assess the impact of prenatal e-cig aerosol exposure on maternal and fetal health, we examined the amino acid signature profiles in maternal and fetal plasma, as well as in the fetal lungs, a sensitive target organ for prenatal tobacco product exposure. Pregnant Sprague Dawley rats were randomly assigned to one of three groups and were exposed to either e-cig aerosols containing nicotine, e-cig aerosols without nicotine, or room air. Dams were exposed utilizing a state-of-the-art custom engineered e-cig vaping system that is compatible with commercially available e-cig atomizers and enables a translational inhalation delivery method comparable to human vaping. Results: We determined that gestational exposure to e-cig aerosols results in significant alterations to the amino acid profile in the maternal and fetal compartments, including the fetal lungs. The data shows a targeted disruption to the nitric oxide pathway, branched-chain amino acid metabolism, fetal protein synthesis, and urea cycle. Conclusion: The data presented herein provides additional support that gestational e-cig aerosol exposure can impact crucial biological processes and exemplifies the need for extensive research on exposure to e-cig aerosols.