We have previously characterized 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) as a non-desensitizing partial agonist at GABA(A) receptors and show that the responses are mediated by short-duration channel openings consonant with single-ligand gated openings of the Cl^- channels. We presently investigated whether responses of cultured rat hippocampal neurones to 4-PIOL are modulated by benzodiazepine (BDZ) and barbiturate receptor ligands. Whole-cell patch-clamp recordings of maximal responses to 1 mM 4-PIOL were comparable in size to responses evoked by 10 μM of the full GABA(A) agonist, isoguvacine. The BDZ receptor inverse agonist, DMCM (1 μM) reduced responses to isoguvacine (to 65.7 ± 11.0%) and 4-PIOL (to 69.3 ± 3.5%) to a similar extent. The BDZ agonist, midazolam (0.1 μM) potentiated responses to both agonists, and resulted in responses with an early peak with later fading. Potentiation of the peak response to 4-PIOL (to 163 ± 14%) was significantly less than for isoguvacine (215 ± 11%). Pentobarbital (50 μM) caused a very marked, but variable, potentiation of the peak response to 4-PIOL (to 484 ± 93%), which was significantly greater than the potentiation of the peak response to isoguvacine (to 304 ± 46%), and induced fading. This suggests that a relatively larger number of the 4-PIOL-induced channel openings can be transformed to longer duration openings by pentobarbital. In conclusion, responses to 4-PIOL and isoguvacine are modulated by BDZ and barbiturate ligands in a qualitatively similar manner, but with a number of quantitative differences which cannot be readily explained by the kinetic model of Macdonald and Twyman (1992). Investigation of these responses at the single-channel level could provide further insight into the operation of the GABA(A) receptor-ionophore complex.