It is well established in the litrature, that selegiline is metabolised to its N-dealkylated metabolites, N-desmethylselegiline, methamphetamine and amphetamine. However, most studies on selegiline metabolism did not characterize the species differences in the formation of the metabolites. Therefore, in this study, we investigated the in vitro metabolism of selegiline in liver microsomes of different species. In addition, to the previously well-characterized metabolites, selegiline-N-oxide (selegiline-NO) was found to be formed as a metabolite of selegiline in rat liver microsomal preparation. The results of experiments with liver microsomes from other species indicated species differences in the rate and extent of formation of selegiline-NO. The dog and hamster liver microsomal preparations were the most active in terms of selegiline-NO production, whereas little selegiline was metabolized to its N-oxide in human liver microsomes. When selegiline-NO was incubated with rat liver microsomes, no metabolism occurred. When a short incubation time was applied in selegiline expriments no increase in the amount of selegiline-NO was detected. Accordingly, it was clear that selegiline was not metabolized to the N-dealkylated or N,N-bis-dealkylated compounds via selegiline-NO. Studies with different isoenzyme inhibitors indicated that the formation of selegiline-NO might be catalyzed at least partly by cytochrome P450 (CYP) 2D6 and CYP3A4. With the exception of hamster microsomes in the microsomal preparations in vitro, the formation of the R,S-stereoisomer of selegiline-NO was preferred.