1. High performance liquid radiochromatographic systems have been developed for the identification and quantification of 7 urinary metabolites of both S-(-)-[3H-N'-CH3]nicotine and R-(+)-[3H-N'-CH3] nicotine in guinea pig, hamster, rat and rabbit. 2. 3'-Hydroxycotinine was a major urinary metabolite of both S-(-)-nicotine and R-(+)-nicotine in guinea pig, hamster and rabbit. Cotinine was not generally a significant urinary metabolite of either nicotine enantiomer, except in rat, where it constituted 14.6 and 10.4%, respectively, of the total radiolabel in the urine after administration of [3H]-S-(-)-nicotine or [3H]-R-(+)-nicotine. Nicotine N'-oxide was an important urinary metabolite of both nicotine isomers in guinea pig and rat, but in both cases, was not observable in hamster and rabbit. No N-methylated urinary metabolite of S-(-)-nicotine could be detected in any of the species examined. In R-(+)-nicotine experiments, only guinea pig afforded N-methylated metabolites. Significant amounts of 2 unidentified polar, non-basic urinary metabolites of both S-(-)- and R-(+)-nicotine-treated animals were observed. 3. Analysis of the comparative metabolism of the nicotine enantiomers in the four animals species studied, showed that stereoselective differences in the formation of oxidative metabolites existed, particularly in the formation of 3'-hydroxycotinine and nicotine-N'-oxide. A clear stereospecificity was observed in the guinea pig, in that only the R-(+)-nicotine enantiomer was N-methylated in this species. 4. Sex differences appear to exist in the metabolism of nicotine enantiomers in the rat. Female rats excreted more of the unidentified polar metabolite B than male rats, whereas the converse was true for nicotine-N'-oxide. In experiments with R-(+)-nicotine, urinary levels of 3'-hydroxycotinine and R-(+)-nicotine in female rats were higher than in male rats. Conversely, higher amounts of nicotine-N'-oxide were observed in the urine of male rats compared to those in female rats.