In this study we determined urine concentration of the tobacco alkaloids anabasine and anatabine, nicotine and its metabolites cotinine, and nornicotine in 99 cigarette smokers and 205 smokeless tobacco users. We also investigated the possibility that anabasine and anatabine can be used as biomarkers for tobacco use during nicotine replacement therapy. Urine samples and data on self-reported tobacco use were obtained from subjects enrolled in tobacco cessation programs. Urine concentrations of tobacco alkaloids and metabolites were measured and correlated with self-reported tobacco use. Concentrations of anabasine and anatabine were used to validate abstinence in smokeless tobacco users who used nicotine gum as part of the therapy. Correlations of alkaloid concentration with self-reported tobacco use before treatment ranged from fair to poor. In subjects abstaining from smokeless tobacco but using nicotine gum, anabasine and anatabine levels were below the cut-point of 2 ng/ml despite high concentrations of nicotine and cotinine resulting from nicotine gum use. Anabasine and anatabine concentrations in urine can be used to validate abstinence or measure the extent of tobacco use in persons undergoing nicotine replacement therapy.