This study investigated the influence of the rate of nicotine metabolism, as indicated by the nicotine metabolite ratio (NMR), on tobacco dependence. We stratified 136 smokers on the basis of saliva NMR as fast (n = 65) and slow (n = 71) metabolizers. Two "loading cigarettes" were smoked after overnight, and a "reward cigarette" was smoked after 6 hours of daytime, abstinence. Blood nicotine concentrations, expired carbon monoxide, withdrawal/craving, and reward questionnaires were collected before/after smoking and during daytime abstinence. Compared with slow metabolizers, fast metabolizers had a shorter nicotine elimination half-life (P < 0.001), lower plasma nicotine concentrations (P < 0.001), and higher withdrawal/craving scores (P < 0.05) for most times during daytime abstinence, indicating that fast metabolizers are likely smoking more to relieve withdrawal symptoms (negative reinforcement). Reward/satisfaction scores were similar in fast and slow metabolizers, suggesting that faster nicotine metabolism, assessed by NMR, is not associated with greater positive reinforcement. CYP2A6 normal (n = 82) and reduced (n = 42) genotype predicted plasma nicotine concentrations but not withdrawal symptoms.Nicotine dependence underlies tobacco dependence and sustains cigarette smoking, which remains a major cause of premature death. 1 Nicotine dependence is motivated by seeking rewarding effects (e.g., stimulation and pleasure), also termed positive reinforcement, and reversing aversive effects of nicotine withdrawal (irritability, anxiety, and difficulty concentrating), also called negative reinforcement.Nicotine is metabolized primarily by the hepatic cytochrome P450 enzyme CYP2A6, with ≈80% of nicotine converted to cotinine (COT), which is further metabolized by the same enzyme to 3′-hydroxycotinine (3HC). 1 There is wide individual variability in the clearance of nicotine, because of both genetic variation and environmental and hormonal factors. The ratio of 3HC/COT, also called the nicotine metabolite ratio (NMR), is a phenotypic