The pharmacological characteristics of digitonin-solubilized peripheral-type benzodiazepine binding sites (PBS) from kidney membranes of various species were investigated to determine whether the species differences and heterogeneity observed in membrane-bound binding sites would be maintained after solubilization. [ 3H]PK 11195 (0.05 to 10 nM) bound with high affinity to rat, guinea pig, calf, and cat kidney solubilized preparations yielding maximal numbers of binding sites ( B max) of 3,593±381, 25,645±1,795, 1,327±141, and 2,446±148 fmol/mg protein, respectively, and equilibrium dissociation constant ( K D ) values of 1.74±0.18, 2.15±0.15, 0.85±0.09, and 1.02±0.06 nM, respectively. On the other hand, the respective B max and K D values for [ 3H]Ro 5-4864 (1.25 to 40 nM) were 2,688±275, 14,182±1,134, 144±23 and 205±17 fmol/mg protein (about 75, 55, 11, and 8%, respectively, of that of [ 3H]PK 11195) and 13.8±1.5, 14.6±1.1, 10.6±1.7, and 19.9±1.2 nM. Unlabeled Ro 5-4864 was two orders of magnitude more potent in displacing [ 3H]PK 11195 binding from rat kidney solubilized preparations than from calf kidney solubilized preparations, whereas the potency of unlabeled PK 11195 in displacing [ 3H]PK 11195 binding from both rat and calf kidney solubilized preparations was almost identical. Analysis of these displacement data revealed that PK 11195 bound to a single population of binding sites ( n H≽1.0 , whereas Ro 5-4864 bound to two populations of binding sites ( n H<1.0) in both rat and calf kidney solubilized preparations. These results indicate that PBS species differences and heterogeneity observed in membrane-bound binding sites are retained in the soluble state and are probably attributable to variations in the molecular structure of PBS rather than to differences in membrane environment.