This study examined whether prepulse inhibition habituates with repeated presentation of the prepulse alone. Prepulse inhibition was determined by measuring the decrement in the startle response when the acoustic startle-eliciting stimulus was preceded by an auditory prepulse. Rats received repetitive exposures of the same auditory prepulse alone (experimental condition) and of a visual prepulse alone (control condition). To reduce habituation of startle itself and the possible dishabituating influence the startle stimulus might have on habituation of prepulse inhibition, startle stimulus presentations were infrequently interspersed among a much larger number of prepulse-alone presentations. Stimulus-specific habituation of prepulse inhibition occurred using an auditory prepulse 2.5 dB, but not 13 dB, above background noise. Implications are discussed for the role of prepulse inhibition in sensory gating.