The in vitro metabolic fate of the anesthetic agent ketamine [(+)2-o-chlorophenyl-2-methylaminocyclohexanone] has been evaluated using microsomal preparations from rat liver. With the aid of a rapid, nonselective metabolite extraction procedure and sample analysis by combined glass capillary gas chromatography low (and high) resolution mass spectrometry, eight metabolites of the drug were identified, six of which have not been reported previously. The novel metabolites were products of alicyclic ring hydroxylation of ketamine and of N-desmethylketamine (norketamine). Semi-quantitative analysis of metabolites produced during microsomal incubation was achieved using glass capillary gas chromatography. The results from this study indicate that 5,6-dehydronorketamine, previously considered to be a major biotransformation product of ketamine in mammalian systems, is almost certainly a methodological artefact.