We utilized cluster analysis to identify individual differences in response to the initial effects of smoking following overnight abstinence among 183 regular smokers. Participants smoked three cigarettes (1 mg nicotine, spaced 30 min apart) in standardized fashion and completed questionnaires about their subjective responses to each cigarette. Heart rate was monitored throughout the procedure. Participants were grouped into two clusters based on their reported subjective effects and heart rate changes to the first cigarette. Clusters differed in terms of greater increases in heart rate, reports of dizziness, sweating, unpleasantness, nausea, and buzzing sensations in one group compared to the other group. The smokers showing increased responses developed greater acute tolerance to the effects of smoking subsequent cigarettes on subjective negative effects and heart rate, and experienced greater negative affect after quitting. These results are partially consistent with a nicotine sensitivity interpretation or a tolerance model of the effects of initial smoking.