Benzodiazepines (BZs) interact with two classes of high affinity binding sites, equilibrium dissociation constants in the nanomolar range, a neuronal or central-type and a non-neuronal or peripheral-type. The peripheral-type binding site has been shown to be present on the outer mitochondrial membrane and appears to be involved in regulation of cholesterol transport in steroid hormone-producing endocrine cells. In rat pituitary GH3 cells, BZs bind to receptors for thyrotropin-releasing hormone (TRH) and via interaction at a different site block Ca2+ influx through voltage-sensitive channels. These, however, are low affinity interactions occurring at micromolar BZ concentrations. Here, using [3H]Ro 5-4864, we report that GH3 cells also have high affinity peripheral-type BZ binding sites. Apparent equilibrium dissociation constants of 7.8 +/- 1.7 nM and 9.3 +/- 4.5 nM for [3H]Ro 5-4864 were measured with intact cells and isolated mitochondria, respectively. As predicted from studies of these sites in other cells, the order of potencies of BZs to displace [3H]Ro 5-4864 was Ro 5-4864 greater than diazepam (DZP) much greater than clonazepam (CIZP); chlordiazepoxide (CDE) did not affect binding. Nifedipine, a dihydropyridine antagonist of Ca2+ channels that has been shown to displace BZs from peripheral-type sites in other cells, was shown to be a competitive inhibitor of [3H]Ro 5-4864 binding with a half-effective concentration in the micromolar range. Ro 5-4864, however, had no effect on Ca2+ influx or efflux in mitochondria isolated from GH3 cells. Hence, GH3 cells exhibit mitochondrial, peripheral-type BZ binding sites but the role of these putative receptors in these neuroendocrine cells, which do not produce steroid hormones, is unclear.