This study examined physiological indicants of the neurobiological mediators of negative affect during acute nicotine withdrawal. Eighty subjects (41 male) were assigned to one of four groups (24-h deprived or nondeprived dependent smokers, occasional smokers, and nonsmokers) and participated in an instructed fear conditioning paradigm involving cued administration of electric shock. Negative affective response was measured with fear-potentiated startle during cues that signaled electric shock and during the postcue offset recovery period. Salivary cortisol and self-report measures were also collected. Fear-potentiated startle results indicated that affective recovery postcue offset was delayed in nicotine-deprived women. Nicotine-deprived women also displayed elevated cortisol levels throughout the fear conditioning procedure.