Aim: N,N-dimethyltryptamine (DMT) is a psychedelic compound under development for the treatment of major depressive disorder (MDD). This study evaluated the in vitro metabolism and clinical pharmacokinetics (PK) of DMT fumarate (SPL026) in healthy subjects. Methods: Results are from the Phase I component of an ongoing Phase I/IIa randomised, double-blind, placebo-controlled, parallel-group, dose-escalation trial. Healthy adults received escalating doses of SPL026 via a 2-phase intravenous (IV) infusion. Dosing regimens were calculated based on PK modelling and predictions, with progression to each subsequent dose level according to safety and tolerability. In vitro experiments assessed hepatic clearance and metabolism of DMT by monoamine oxidase (MAO) and cytochrome P450 enzymes. Results: 24 healthy subjects received escalating doses of SPL026 which were safe and well-tolerated. Dose-proportional increases in DMT exposure were observed over the range of 9–21.5 mg. For all doses, median time to peak plasma concentration was ~10 min and mean elimination half-life was 9–12 min. There was no relationship between peak DMT plasma concentration and body mass index, weight or age. In vitro hepatic mitochondrial fraction clearance of SPL026 was inhibited by MAO-A, but not MAO-B, inhibition. CYP2D6 and CYP2C19 modified SPL026 clearance in vitro. The unbound fraction of SPL026 was approximately 70%. Conclusion: This is the first study to determine, in detail, the full PK profile of DMT in humans, confirming rapid attainment of peak plasma concentrations followed by accelerated clearance. These findings provide evidence which support the development of novel DMT infusion regimens for the treatment of MDD.