A detailed study of the urinary metabolites of N'-nitrosonornicotine has been perfomed, employing a simple high pressure liquid chromatographic method. The percentage excretion of the principal urinary metabolites was determined over a dose range of 3-300 mg/kg in the F-344 rat, as follows: 4-hydroxy-4-(3-pyridyl)butyric acid (37.1-53.3%, respectively, of the dose), N'-nitrosonornicotine-l-N-oxide (6.7-10.7%), norcotinine (3.2-5.1%), 4-oxo-4-(3-pyridyl)butyric acid (31.1-12.8%), N' -nitrosonornicotine (3.3-5.2%). In the strain A mouse and Syrian golden hamster, the urinary metabolites were qualitatively similar to those observed in the F-344 rat. The interrelationships of the various metabolites of N'-nitrosonornicotine which have been observed in vitro and in vivo were established. The in vitro metabolites resulting from 2'-hydroxylation by liver microsomes, myosmine and 4-hydroxy-l-(3-pyridyl)-1-butanone were converted, by the F-344 rat, primarily to 4-oxo-4-(3-pyridyl)butyric acid as a urinary metabolite. The in vitro metabolite resulting from 5'-hydroxylation by liver microsomes, 2-hydroxy-5-(3-pyridyl)tetrahydrofuran, gave 4-hydroxy-4-(3-pyridyl)butyric acid as its major urinary metabolite, apparently via 5-(3-pyridyl)-tetrahydrofuran-2-one. N'-nitrosonornicotine-l-N-oxide, the remaining major in vitro metabolite, was excreted to a large extent unchanged in F-344 rat urine. The urinary metabolites from 2'-hydroxylation and 5'-hydroxylation of N'-nitrosonornicotine, 4-oxo-4-(3-pyridyl)butyric acid and 4-hydroxy-4-(3-pyridyl)butyric acid, respectively, were not formed from the in vivo metabolite norcotinine and were not interconverted significantly by the F-344 rat. Thus, these metabolites appear to be reliable indicators for the two possible in vivo α-hydroxylations of N'-nitrosonornicotine.