In this study we examined the binding characteristics of the gamma-aminobutyric acid (GABAA) receptor complex after chronic pentobarbital sodium treatment in cultured mammalian cortical neurons. Chronic pentobarbital sodium treatment (200 microM, 5 days) did not alter the basal binding of ligands like [3H]flunitrazepam, [3H]ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5 alpha][1,4]-BZ-3-carboxylate and [3H]ethyl-8-azido-5,6-dihydro- 5-methyl-6-oxo-4H-imidazo [1,5 alpha][1,4]BZ-3-carboxylate that bind to the benzodiazepine (BZ) recognition site of the GABAA receptor complex. Similarly, chronic pentobarbital sodium treatment did not alter the basal binding of [3H]GABA and t-butylbicyclophosphoro[35S]thionate. However, chronic pentobarbital sodium treatment produced uncoupling between GABA, barbiturate and neurosteroid sites with the BZ site. The efficacy (Emax) values of GABA, pentobarbital and neurosteroid, 5 alpha-pregnan-3 alpha-ol-20-one, on [3H]flunitrazepam binding were significantly decreased, whereas their potency (EC50) values were not altered after chronic pentobarbital sodium treatment. Taken together, these results suggest that chronic pentobarbital sodium treatment produces heterologous uncoupling of the GABA-BZ receptor ionophore complex.