A technique was described for the measurement of specific [3H]diazepam [a CNS depressant] binding to subcellular fractions of rat brain. Binding occurred in fresh or hypotonically shocked, frozen and thawed preparations. Binding was independent of the buffer used and was concentrated in the synaptosomal fraction. The total binding capacity to crude brain homogenate was entirely recovered in the particulate fractions (P1, P2 and P3). Binding was saturable and reached 34 pmol/g of tissue. Half-maximal binding (KD) occurred at 3.2 nM. Hill and Scatchard analysis indicated that the binding was noncooperative and to a single class of sites. Binding was time dependent and reversible. The bimolecular association constant (K1) was 1.13 .cntdot. 106 s-1 M-1 and the 1st order dissociation constant (K-1) was 2.69 .cntdot. 10-3 s-1. Binding was highest in cerebral cortex, hippocampus and cerebellum; intermediate in midbrain, hypothalamus, corpus striatum and medulla oblongata/pons; and lowest in spinal cord. Benzodiazepines inhibited binding of [3H]diazepam in a manner correlated with pharmacological activity in vivo. Binding was not inhibited by non-benzodiazepine anxiolytics, muscle relaxants, anticonvulsants or by strychnine and glycine. Distributin of [3H] diazepam binding in several regions of the rat, CNS correlated with Na+-independent binding of GABA in the same regions. GABA was possibly involved in the mechanism of action of the benzodiazepines. A mechanism based on the interaction of benzodiazepines with central glycine receptors was not supported.