Background: Cotinine, a nicotine metabolite, is a biomarker of tobacco, nicotine, and carcinogen exposure. However, a given cotinine level may not represent the same tobacco exposure; for example, African-Americans have higher cotinine levels than Caucasians after controlling for exposure. Methods: Cotinine levels are determined by the amount of cotinine formation and the rate of cotinine removal, which are both mediated by the enzyme CYP2A6. Because CYP2A6 activity differs by sex (estrogen induces CYP2A6) and genotype, their effect on cotinine formation and removal was measured in nonsmoking Caucasians (Study 1, n 181) infused with labeled nicotine and cotinine. The findings were then extended to ad libitum smokers (Study 2, n 163). Results: Study 1: Reduced CYP2A6 activity altered cotinine formation less than cotinine removal resulting in ratios of formation to removal of 1.31 and 1.12 in CYP2A6 reduced and normal metabolizers (P 0.01), or 1.39 and 1.12 in males and females (P 0.001), suggesting an overestimation of tobacco exposure in slower metabolizers. Study 2: Cotinine again overestimated tobacco and carcinogen exposure by 25% or more in CYP2A6 reduced metabolizers (2-fold between some genotypes) and in males. Conclusions: In people with slower relative to faster CYP2A6 activity, cotinine accumulates resulting in substantial differences in cotinine levels for a given tobacco exposure. Impact: Cotinine levels may be misleading when comparing those with differing CYP2A6 genotypes within a race, between races with differing frequencies of CYP2A6 gene variants (i.e., African-Americans have higher frequencies of reduced function variants contributing to their higher cotinine levels), or between the sexes.