The properties of [3H]clonazepam, [3H]diazepam and [3H]zolpidem (N,N,6[trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitratrate) binding to synaptic membranes of cerebellum, cortex, olfactory bulb, striatum and spinal cord of rat were compared to the binding properties of [3H]flunitrazepam, [3H]flumazenil and [3H]midazolam. In the cerebellar, cortical and olfactory bulb membranes, the density of high-affinity binding sites of all these tritiated benzodiazepine (BZ) ligands is almost identical. In contrast, in the striatum, the density of [3H]clonazepam and [3H]zolpidem binding sites is approximately 60 and 30%, respectively, of the density of [3H]diazepam, [3H]flunitrazepam or [3H]flumazenil sites. In spinal cord membranes, the number of high-affinity binding sites of [3H]clonazepam and [3H]zolpidem is less than 20% of the number of binding sites for [3H]diazepam, [3H]flunitrazepam, [3H]flumazenil and [3H]midazolam. Moreover, the displacement of [3H]flunitrazepam from spinal cord membranes by clonazepam and zolpidem was characterized by high IC50 values and Hill slopes significantly less than 1. Because [3H]BZ ligand binding in the spinal cord is enhanced by gamma-aminobutyric acid (GABA), these data suggest that different regions of the rat central nervous system may contain different GABA-BZ receptor subtypes. The different pharmacological properties of clonazepam, diazepam and zolpidem (i.e., regarding their ability to enhance bicuculline seizure threshold, to decrease locomotor activity, to induce ataxia or to elicit anticonflict action) further support the concept that in the rat central nervous system preferential occupancy of heterogeneous GABAA receptors by these drugs can be related to their effects on behavior.