Experiments were performed to characterize [35S]TBPS binding in rat cortical synaptoneurosomes, which have vesicular structures containing both pre- and postsynaptic elements. Scatchard analysis revealed a single component of [35S]TBPS binding sites with KD and Bmax values of 76.1 nM and 1.97 pmoles/mg protein, respectively, under physiological conditions. GABA and muscimol inhibited [35S]TBPS binding in a concentration-dependent manner. IC50 values of these GABAA agonists in displacing synaptoneurosomal [35S]TBPS binding are comparable to previously reported EC50 values of the agonist-stimulated 36Cl- uptake in synaptoneurosomes by these agents. Furthermore, the IC50 values of these GABAA agonists were better correspondence to those determined by [3H]muscimol binding in synaptoneurosomal preparations as reported by Delorey and Brown (3) than those determined in membrane preparations. Although bicuculline increased [35S]TBPS binding in a concentration dependent manner in cortical membranes, it did not affect synaptoneurosomal [35S]TBPS binding. Benzodiazepine agonists and inverse agonists (0.1 to 10 microM) did not show any effects on the binding in the absence of muscimol. However, benzodiazepine agonists potentiated and inverse agonists antagonized muscimol-induced inhibition of synaptoneurosomal [35S]TBPS binding. In addition, an anesthetic steroid, THDOC, and pentobarbital inhibited synaptoneurosomal [35S]TBPS binding in a concentration dependent manner. These results suggest that allosteric modulation of [35S]TBPS binding by various ligands which interact with GABAA supramolecular complexes remain intact in synaptoneurosomes. It appears that this preparation is useful for investigating correlation between functional 36Cl- uptake and individual binding studies of each of the GABAA receptor complex.