Background: Rodatristat ethyl (RE) targets peripheral serotonin (5HT) biosynthesis and is in Phase IIb development (ELEVATE 2) for pulmonary arterial hypertension (PAH). Upregulated 5HT signaling in lung drives pulmonary artery smooth muscle cell proliferation and vascular remodeling in PAH. Here, in healthy subjects, we characterize the magnitude and time to maximal reductions in peripheral 5HT biosynthesis to levels associated with efficacy in nonclinical PAH models, and the reversibility following RE withdrawal. Methods: Healthy subjects (n=79) received 100-800 mg BID of RE over 14-16 days. 5HT reductions were assessed via plasma and urine 5-HIAA (biomarker of 5HT levels) at baseline, last day of treatment and 7 days later. Results: Maximal 5-HIAA reductions in plasma and urine were achieved by 7 days of RE treatment. Reductions in plasma and urine 5-HIAA were correlated (Pearson R=0.5514; p<0.05). RE ? 300 mg BID led to a dose proportional decrease in plasma and urine 5-HIAA (-46% plasma; -47% urine at 300 mg BID). Doses >300 mg BID yielded only a further ~10% reduction in 5-HIAA (-54% plasma; -59%, urine at 800 mg BID). Doses of ?300 mg BID achieved ~40% reduction in urine 5-HIAA associated with efficacy in rat monocrotaline and SUGEN hypoxia PAH models. Inhibition of 5HT biosynthesis was reversible as 5-HIAA returned to near baseline values by 7 days post dosing. Conclusions: RE achieved dose-dependent reductions in serotonin synthesis in healthy subjects. A target ~40% reduction associated with efficacy in rat PAH models was achieved by day 7 for RE doses ?300mg BID. Inhibition of 5HT biosynthesis was reversible.