Men and women may differ in their sensitivity to the mood-modulating properties of nicotine. Male and female adult smokers were exposed to four sessions crossing two nicotine deprivation conditions (12-h deprived vs. nondeprived) with two drug conditions (nicotine vs. placebo nasal spray). Acoustic probes elicited startle eyeblink responses while viewing affective and cigarette-related slides. In-session mood ratings were collected to gauge self-reported negative affect, positive affect, and craving. Nicotine nasal spray reduced startle amplitude in both men and women following 12-h deprivation compared with smoking nondeprivation. During nondeprivation, nicotine nasal spray increased startle amplitude in women compared with placebo spray, whereas no difference was found for men. The startle results suggest that both men and women are responsive to the hedonic properties of nicotine.