In mammalian brain peripheral benzodiazepine (PBZD) receptors are predominantly localized on astroglial cells. Previous studies utilizing whole membrane preparations from brain and peripheral organs of various species have indicated several distinctions between the drug-receptor interactions of the two prototypic PBZD receptor ligands, PK 11195 and Ro5-4864. The present study was undertaken t o determine whether putative differences in the binding of PBZD receptor ligands in homogenates of primary astrocyte cultures can be interpreted as the labeling of PBZD receptor subtypes. Equilibrium competition and saturation binding experiments in homogenate preparations of primary astrocytes from cerebral cortex of new born rats revealed that L3H]PK 11195 labels twice the number of [3H]Ro5-4864 binding sites. Unlabeled Ro5-4864 competes for 13HJPK 11195 binding in a manner suggesting the existence of multiple PK 11195 binding sites. The competition binding experiments, using various benzodiazepines, indicate that one binding component of PK 11195 corresponds t o Ro5-4864 binding sites, whereas the second is different. The latter binding site does not correspond to the central BZD receptor but displays the pharmacological properties of the PBZD receptor. Further differences between the binding of PK 11195 and Ro5-4864 in astrocytes were detected in the presence of ethanol which was more effective in inhibiting the binding of the latter. Subcellular distribution studies indicated, however, that the binding of both [3H]PK 11195 and r3H]Ro5-4864 is associated primarily with the mitochondria1 fraction of astrocytes. Taken together, the present study indicates the existence of non-overlapping PBZD binding sites in astrocytes and thus suggests the existence of PBZD receptor subtypes. It appears, however, that there is no distinction in the subcellular localization of the putative subtypes of the PBZD receptor. o 1993 Wiley-Liss, Inc. 0 1993 Wiley-Liss, Inc (PBZD) receptor has been detected in several tissues, such as heart, lung, kidney, and testis, and also in mammalian brain (Marangos et al., 1982). The PBZD receptor is not associated with the GABA receptor complex and its precise physiological significance is not entirely clear. Evidence suggests, however, that the PBZD re-