Mammals metabolize the tobacco alkaloid (S)-nicotine primarily to the lactam (S)-cotinine by a pathway involving an initial cytochrome P-450 catalyzed two-electron oxidation at the prochiral 5'-carbon atom. The stereochemical course of this oxidation was examined with human microsomal preparations and the E and Z diastereomers of (S)-nicotine-5'-d1. The metabolically generated A'(5'-iminium ion intermediate was trapped and analyzed as the corresponding diastereomeric 5'-cyano derivatives by a capillary column GC-EIMS selected ion monitoring assay. The results of these studies established that this biotransformation proceeds with the stereoselective abstraction of the 5'-pro-E proton, that is, the C-5' proton trans to the bulky pyridyl group. The observed stereoselectivity was independent of proton vs. deuteron abstraction. Additionally, the extent of (S)-cotinine formation was minor and did not influence the stereochemical composition of the metabolically derived a-cyano amines. Studies with male Dutch rabbit liver microsomal preparations gave similar results. These findings suggest that the structure of the complex formed between (S)-nicotine and the active site of cytochrome P-450 is highly ordered and dictates the stereochemical course of the reaction pathway.