Background: Smokeless tobacco is an efficient delivery vehicle for nicotine and can contain significant amounts of carcinogens. However, few studies have examined factors that might moderate levels of nicotine or carcinogen exposure. Aims: To determine the effect of duration of smokeless tobacco use on the uptake of nicotine and a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK). Methods: Questionnaires on use of smokeless tobacco were administered, and urine samples from 212 smokeless tobacco users were analysed for biomarkers of uptake of nicotine and NNK. The biomarkers were cotinine and total 4-(methylnitrosamino)-1-(3-pyhdyl)-1-butanol (NNAL). Male smokeless tobacco users were recruited for studies designed to investigate methods of reducing smokeless tobacco use. The questionnaire and biomarker data were obtained at baseline, prior to reduction. Results: Levels of cotinine (p<0.001) and total NNAL (p<0.001) were significantly correlated with duration (in years) of use of smokeless tobacco products. Median cotinine and total NNAL were 2.4 and 2.1 times higher, respectively, in the ≥21 years of use than in the 0-5 years of use category. Conclusions: Smokeless tobacco users adjust their intensity of use with experience in order to increase their nicotine dose, resulting in a corresponding increase in exposure to NNK, a powerful carcinogen. These results indicate the importance of educating smokeless tobacco users about the effects of prolonged use of these products.