The carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are believed to play a role in cancers associated with the use of tobacco products. Urinary metabolites of NNK and NNN could be used as biomarkers for an individual's ability to metabolically activate or detoxify these nitrosamines. While several metabolites of NNK can be quantified in human urine, no assay is available to determine human urinary levels of NNK and NNN metabolites resulting from the critical alpha-hydroxylation metabolic activation pathways. The major urinary metabolites resulting from alpha-hydroxylation of NNK and NNN in rodents are 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). The major obstacle to the use of these metabolites as biomarkers of metabolic activation is the fact that they are also metabolites of nicotine, which is present at levels 1400-13000 times greater than those of the nitrosamines in cigarette smoke. However, the chirality of hydroxy acid could be useful in overcoming this problem. If different enantiomers of hydroxy acid were formed from nicotine versus the nitrosamines, and if the overall yield of hydroxy acid from nicotine were substantially smaller than that from the nitrosamines, then hydroxy acid might be useful as a urinary biomarker of NNK and NNN alpha-hydroxylation. To these ends, F-344 rats were administered either [5-3H]NNK, [5-3H]NNN, [5-3H]keto acid, or [2'-14C]nicotine. The levels of urinary hydroxy acid were determined by HPLC analysis. Its stereochemistry was determined by conversion to its methyl ester, reaction with (S)-(-)-alpha-methylbenzyl isocyanate, and separation and quantitation of the resulting diastereomers by HPLC. Urinary hydroxy acid accounted for 12% of the NNK dose and 31% of the NNN dose, but only 1 and 0.1% of the dose of keto acid and nicotine, respectively. Furthermore, metabolism of NNK produced mainly (S)-hydroxy acid in the urine, while metabolism of keto acid and nicotine gave predominantly (R)-hydroxy acid. Both enantiomers were present in the urine of NNN-treated rats. Therefore, in the rat, it is possible to distinguish the hydroxy acid derived from nicotine from that derived from the nitrosamines. If similar pathways occur in humans, (S)-hydroxy acid could potentially be developed as a urinary biomarker of NNK and NNN alpha-hydroxylation in smokers.