A new radioimmulWassay sufficiently sensitive and specific to permit estimation of nicotine in human plasma in the presence of its principal metabolite, cotinine, is described. Plasma nicotine concentrations determined after inhalation of ten puffs of cigarette smoke were higher for each of seven time points in habituated than in naive smokers. Mean nicotine concentrations of 6 habituated and 6 naive smokers one minute after cessation of smoking were 20.6 ± 3.4 and 7.6 ± 2.9 ng/ml, respectively. Studies on buccal absorption of nicotine in volunteers who smoked 10 puffs, holding each puff in the mouth for 10 seconds without inhalation of smoke, revealed similar plasma nicotine concentrations one minute after smoking of 1.4 ± 0.4 and 1.2 ± 0.2 ng/ml for habituated and naive smokers, respectively. Blank values for nicotine in each subject were obtained prior to smoking and subtracted from subsequent plasma nicotine concentrations. Means of blank values for nicotine in fasted naive and habituated smokers who refrained from smoking at least 8 hours prior to the experiments were 4.5 ± 1.3 and 3.8 ± 0.7 ng/ml, respectively.