Previous research with both animal and human subjects has shown that startle reflex magnitude is potentiated in an aversive stimulus context, relative to responses elicited in a neutral or appetitive context. In the present experiment, the same pleasant, unpleasant, and neutral picture stimuli were repeatedly presented to human subjects. Startle reflex habituation was assessed in each stimulus context and was compared with the habituation patterns of heart rate, electrodermal, and facial corrugator muscle responses. All systems showed initial differentiation among affective picture contents and general habituation over trials. The startle reflex alone, however, continued to differentiate among pleasant, neutral, and unpleasant pictures throughout the presentation series. These results suggest that (a) the startle probe reflex is relatively uninfluenced by stimulus novelty, (b) the startle modulatory circuit (identified with amygdala-reticular connections in animals) varies systematically with affective valence, and (c) the modulatory influence is less subject to habituation than is the obligatory startle pathway or responses in other somatic and autonomic systems.