The metabolism of α,α,β,β-tetradeutero-N,N-dimethyltryptamine (D4DMT) in rat brain in vivo as a function of time and dose was examined. Quantification of D4DMT and its respective deuterometabolites was accomplished using gas chromatographic/mass spectrometric/selected ion monitoring/isotope dilution techniques. The results of this study indicate that D4DMT is metabolized to the corresponding deutero-N-methyltryptamine, tryptamine, 1,2,3,4-tetrahydro-beta-carboline, and 2-methyl-1,2,3,4-tetrahydro-beta-carboline in rat brain. The subcellular distribution of D4DMT and the aforementioned metabolites is also reported.