Recent data from nicotine-dependent rats (14) and healthy smokers (18) would suggest that nicotine withdrawal modulates the acoustic startle reflex in a way similar to that of fear (5,20). We examined this directly using nonsmokers and healthy smokers who had no deprivation, brief deprivation (2-3 h), or prolonged deprivation (15 h). Groups differences in heart rate (HR), alveolar carbon monoxide (CO) levels, and desire and craving for cigarettes confirmed the presence of smoking withdrawal. However, there were no significant differences in the magnitude of the baseline startle response among the differently deprived smokers or between the smokers and the nonsmokers. Subsequent startle tests were carried out in the smokers during repeated sequences of preparing a cigarette for smoking (smoking cues) and then smoking. Whereas we did find statistically significant interactions of smoking deprivation with smoking cues and with renewed smoking, there was only weak confirmation of a priori predictions of motivational effects of smoking cues or of smoking. We conclude that smoking dependence may not affect the acoustic startle response itself; modulation seems to occur, but only after experience with the test situation. Discussed were possible mechanisms of this modulation in both humans and animals and further application of the startle response for providing interdisciplinary assessments of the motivational effects of nicotine withdrawal.