Negative affect is thought to be an important factor in the maintenance of cigarette smoking, and thus it is important to further develop objective measures of smoking-related emotional responses. Nonsmokers, nonabstinent smokers, and abstinent smokers participated in a cue reactivity task where eyeblink startle amplitude and startle probe P300 (P3) suppression were measured during the presentation of emotional pictures. During unpleasant pictures, the amplitude of both measures was smaller in nonabstinent smokers than in nonsmokers or abstinent smokers. P3 suppression, but not startle amplitude, was larger in abstinent smokers than in nonsmokers. Abstinence-induced increases in cigarette craving were associated with P3 suppression during tobacco-related pictures. Results suggest that tobacco abstinence increases emotional reactivity to unpleasant stimuli, which is consistent with negative reinforcement models of tobacco addiction.