People who use tobacco products are exposed to considerable amounts of N'-nitrosonornicotine (NNN), a well-established esophageal carcinogen in rats. NNN is believed to play a significant role as a cause of esophageal and oral cavity cancer in smokers and snuff dippers. The carcinogenicity of NNN is dependent on its metabolic activation. However, virtually all studies carried out to date on NNN metabolism have used racemic material. In this study, we examined the metabolism of [5-(3)H]-(S)-NNN and [5-(3)H]-(R)-NNN in cultured rat esophagus and in vivo in rats. Cultured rat esophagus metabolized (S)-NNN (1 microM) predominantly to products of 2'-hydroxylation, 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol). In contrast, the major metabolite of (R)-NNN under these conditions was 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), a product of NNN 5'-hydroxylation. The 2'-hydroxylation:5'-hydroxylation metabolite ratio ranged from 6.22 to 8.06 at various time intervals in the incubations with (S)-NNN, while the corresponding ratios were 1.12-1.33 in the experiments with (R)-NNN. These differences were statistically significant (P<0.001). Since 2'-hydroxylation is believed to be the major metabolic activation pathway of NNN in the rat esophagus, the results demonstrate that (S)-NNN is metabolically activated more extensively than (R)-NNN in this tissue, and therefore may be more carcinogenic. Rats were treated with 0.3 mg/kg of [5-(3)H]-(R)-NNN, [5-(3)H]-(S)-NNN, or racemic [5-(3)H]NNN by gavage, and the urinary metabolites were analyzed. The major metabolites were hydroxy acid and keto acid. As in the in vitro studies, products of 2'-hydroxylation predominated in the urine of the rats treated with (S)-NNN while products of 5'-hydroxylation were more prevalent in the rats treated with (R)-NNN. 2'-Hydroxylation:5'-hydroxylation metabolite ratios ranged from 1.66 to 2.04 in the urine at various times after treatment with (S)-NNN, while the ratios were 0.398-0.450 for the rats treated with (R)-NNN (P<0.001). The results of this study provide new insights into NNN metabolism in rats and suggest that the carcinogenicity of (S)-NNN, the predominant enantiomer in tobacco products, may be greater than that of (R)-NNN or racemic NNN.