Theories of habituation have included an increase in postsynaptic inhibition as one possible mechanism underlying response decrement following repetitive stimulus presentation. In this study, the glycine antagonist strychnine (1.0 mg/kg, ip, 10 min pretreatment) was used to investigate the involvement of glycinergic neurons in the development and/or expression of short-term (within-session) habituation (Experiment 1) and long-term (between-sessions) habituation (Experiments 2 and 3) of the acoustic startle response in rats. Over a range of eliciting-stimulus intensities (95, 105, and 115 dB) and interstimulus intervals (3, 7, 13, and 27 s), strychnine markedly increased startle amplitude, relative to water injection, whereas it failed to attenuate the rate of within-session habituation (Experiment 1). In Experiment 2, rats that were exposed to daily sessions of startle-eliciting stimuli for 4 days and then tested on the fifth day showed lower overall levels of startle amplitude, relative to rats that had not received prior habituation training. Strychnine injected prior to the test session again increased startle amplitude but did not block the expression of between-sessions habituation. In Experiment 3, rats that were injected with either strychnine or water prior to each of three daily habituation training sessions and subsequently tested on Day 4 showed similar between-sessions habituation, relative to untrained rats that had received daily injections in the animal room. In summary, strychnine increased startle amplitude without affecting either within-session or between-sessions habituation of acoustic startle. These results emphasize the need to discern between drug effects on response amplitude per se and effects on response habituation. Furthermore, the data indicate that a buildup of inhibition in glycinergic neurons does not explain either within-session or between-sessions habituation of acoustic startle in rats. These results are discussed in light of current theories of habituation.