BACKGROUND: Accurate measurement tools of exposure for use in large epidemiologic studies are lacking. Biomarkers of tobacco exposure provide additional advantages to self-reports and there is a need to further develop and validate them. The objective is to compare toenail nicotine levels, a novel biomarker of tobacco exposure, with self-reports of tobacco exposure from a large cohort study. METHODS: In this cross-sectional analysis, toenail samples were collected from 2,485 women participating in the Nurses' Health Study in 1982. Detailed self-reports of smoking habits and reported exposure to secondhand smoke (SHS) were collected from these women near the time of toenail collection. The toenail samples were analyzed by a high-performance liquid chromatography method for measuring nicotine. RESULTS: The 5 to 95 percentile range of toenail nicotine was from 0.06 to 4.06 ng/mg toenail and the median level was 0.21 ng/mg. There was a significant difference in toenail nicotine levels according to reported smoking status (the median level for nonsmokers with no SHS was 0.10 ng/mg, the median level for nonsmokers with SHS was 0.14 ng/mg, and the median level for active smokers was 1.77 ng/mg). However there was considerable overlap in nicotine levels according to reported smoking status. Toenail nicotine level was strongly associated with reported smoking level (Spearman r = 0.63), but there was no complete concordance, suggesting that the two methods are measuring different aspects of the same exposure. CONCLUSION: Our findings show that toenail nicotine levels capture the overall burden of tobacco smoke exposure and provide additional information on exposure not captured by reported history.