N′-Nitrosonornicotine (NNN) is present in significant quantities in tobacco and tobacco smoke and is believed to play an important role as a cause of cancer in people who use tobacco products. Biomarkers of NNN uptake in humans such as urinary metabolites would be useful for assessing cancer risk. Previous studies, carried out almost exclusively in rodents, have characterized urinary metabolites of NNN, but none of these would be suitable as a biomarkers of NNN uptake in humans. Therefore, we studied NNN metabolism in the patas monkey. Monkeys were treated intravenously with [5-3H]NNN, which has tritium in the pyridine ring. Blood and urine samples were collected at timed intervals. Six urinary metabolites were observed by high-performance liquid chromatography (HPLC) and were identified by their spectral properties and/or comparison to appropriate standards as follows: metabolite (% of radioactivity eluting from HPLC ± S.D., n = 3 monkeys); 4-hydroxy-4-(3-pyridyl)butyric acid (43.8 ± 4.0); 4-oxo-4-(3-pyridyl) butyric acid (2.7 ± 0.66); norcotinine (13.1 ± 2.7); norcotinine-1N-oxide (16.5 ± 1.3); 3′-hydroxynorcotinine (16.9 ± 2.0); 3′-(O-β-D-glucopyranuronosyl)hydroxynorcotinine (5.4 ± 1.0); and unchanged NNN (0.63 ± 0.15). The two major metabolites in serum were 4-hydroxy-4-(3-pyridyl)butyric acid and norcotinine. NNN was rapidly metabolized to 4-hydroxy-4-(3-pyridyl)butyric acid, whereas the formation of norcotinine and 3′-hydroxynorcotinine were somewhat delayed. The results of this study demonstrate substantial differences between NNN metabolism in the rodent and patas monkey. Metabolism of NNN to norcotinine and its derivatives was far more prevalent in the patas monkey than in the rat. 3′-Hydroxynorcotinine and its O-glucuronide may be formed from NNN via α-oximinonornicotine or isomyosmine. There was no evidence that it was formed via norcotinine, although this pathway could not be excluded. 3′-Hydroxynorcotinine could potentially be a biomarker of NNN uptake in humans.