Two experiments examined the frequency specificity of habituation of the acoustic startle response in the rat. Following the long-term habituation of startle to one of two pure tone stimuli in Experiment 1, animals were presented with the other stimulus. Startle response asymptotes were unaffected by this change in stimulus frequency. Short-term habituation of startle also was insensitive to stimulus frequency. In Experiment 2, pure tone stimuli were used to provoke both a startle response and the interruption of drinking. Long-term habituation of startle to either stimulus was unaffected by a change in frequency. Animals that received the two stimuli on alternating days showed as rapid a habituation as did the groups receiving only one stimulus frequency during acquisition. Conversely, the lick suppression measure was found to be frequency specific. Lick suppression durations rose to pre-habituation levels when the frequency of the stimulus was changed. Animals that received the two stimuli on alternating days showed retarded habituation compared to those groups presented with only one stimulus frequency during acquisition. Although long-term habituation of startle is not stimulus specific, it is mediated by central processes and thus remains a valuable model in the study of neurophysiological mechanisms of behavioral change.