The acoustic startle reflex is strongly inhibited by a moderate-intensity acoustic stimulus that precedes the startling stimulus by roughly 10–1000 ms (prepulse inhibition, PPI). At long interstimulus intervals (ISIs) of 100–1000 ms, PPI in rats is reduced by the muscarinic receptor antagonist scopolamine. Here, we studied the role of GABA receptors in PPI at full ISI ranges in both mice and rats. In B6 mice, PPI begins and ends at shorter ISIs (4 and 1000 ms, respectively) than in Wistar rats (8 and 5000 ms). The GABAA antagonist bicuculline (1 mg/kg i.p.) reduced PPI at ISIs near the peak of PPI in both rats and mice. The GABAB antagonist phaclofen (10 or 30 mg/kg i.p. in rats or mice, respectively) reduced PPI only at long ISIs, similar to the effects of the muscarinic antagonist scopolamine (1 mg/kg i.p.). The effects of phaclofen and scopolamine were additive in rats, suggesting independent effects of GABAB and muscarinic receptors. Patch-clamp recordings of startle-mediating PnC (nucleus reticularis pontis caudalis) giant neurons in rat slices show that EPSCs evoked by either trigeminal or auditory fiber stimulation were inhibited by the GABAA/C agonist muscimol or the GABAB agonist baclofen via postsynaptic mechanisms. Hyperpolarization of PnC neurons by muscimol was reversed with bicuculline, indicating that postsynaptic GABAA receptors strongly inhibit PnC giant neurons needed for startle. Therefore, GABA receptors on PnC giant neurons mediate a substantial part of PPI, with GABAA receptors contributing at the peak of PPI, and GABAB receptors adding to muscarinic effects on PPI at long ISIs.