Specific biomarkers of tobacco carcinogen uptake are critical for investigations of the role of tobacco smoke exposure in human cancers. Two new biomarkers of human exposure to tobacco-specific carcinogens have been recently developed by our research group: urinary N¶-nitrosonornicotine (NNN) and toenail 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In this study, we report the presence of NNN in human toenails. Toenails of 17 smokers were analyzed for total NNN. Mean total NNN level in these samples was 4.63 ± 6.48 fmol/mg toenail and correlated with previously reported total NNAL (r = 0.96; P < 0.0001), total nicotine (r = 0.48; P < 0.05), and total cotinine (r = 0.87; P < 0.0001). An interesting finding was that amounts of NNN in smokers' toenails were generally higher than those of total NNAL. The ratio of toenail NNN to NNAL averaged 2.8, whereas the previously reported ratio between these biomarkers in smokers' urine was 0.1. NNN was also found in toenail samples from 12 nonsmokers, averaging 0.35 ± 0.16 fmol/mg and positively correlating with toenail cotinine (r = 0.58; P = 0.05). The results of this study show the feasibility of quantifying NNN in human toenails, providing a potentially useful new biomarker of tobacco carcinogen exposure.