Cultures of cerebral neurons were prepared from chick embryos, 8.5 days in ovo, and maintained in vitro. Following chronic exposure of these cells to GABA, the levels of [3H]flunitrazepam binding in situ and electrophysiological responsiveness to γ-aminobutyric acid (GABA) was examined. Treatment with 100 μM GABA for 7 days reduced [3H]flunitrazepam binding in situ by 70 ± 8% compared to untreated controls. The binding of [3H]N-methylscopolamine was unaffected by this treatment. The reduction in [3H]flunitrazepam binding was prevented by concomitant exposure of developing neurones to the GABA antagonist R 5135, suggesting that GABAA receptor occupancy is required. The loss of bezodiazepine receptors was dependent on the GABA concentration in the culture medium and a half-saturation (IC50) value of 11.2 ± 3.7 μM was estimated. Whole-cell patch-clamp recordings were obtained to assess the functional properties of the labile receptor pool observed in the binding studies. Neurons cultured with 100 μM GABA for 7 days showed a 60-70% reduction in the peak current amplitudes observed in response to application of 10-100 μM GABA. However, the rate of rapid desensitization, quantified by measuring changes in input conductance, was unchanged by chronic GABA exposure, yielding decay time constants of 27.1 ± 2.1 and 34.7 ± 4.7 s for control and treated cells, respectively. The results are consistent with a GABA modulation of the GABAA/benzodiazepine receptor complex by means of down-regulation. © 1989.