Benzodiazepines (BZs) have been shown to modulate voltage-sensitive Ca2+ channels in a number of neuronal and nonneuronal cell types and to competitively antagonize TRH binding to receptors on cells of the nervous system and anterior pituitary gland. Because interaction of TRH with its receptor is known to cause enhanced influx of Ca2+ through voltage-sensitive channels in rat pituitary GH3 cells, it was determined whether BZs and TRH were interacting with the same binding site on these cells. The potencies of three BZs, Ro5-4864, diazepam (DZP), and chlordiazepoxide (CDE), were compared as modulators of Ca2+ channels and as inhibitors of TRH binding in GH3 cells. Modulation of Ca2+ channel activity was measured as the inhibition by BZs of K+ depolarization-induced Ca2+ influx using intracellularly trapped quin 2 or 45Ca2+ uptake. The three BZs caused dose-dependent inhibition of Ca2+ influx with an order of potency of Ro 5-4864 greater than DZP greater than CDE. In contrast, the order of potency of the three BZs to inhibit [3H]TRH binding was CDE greater than DZP much greater than Ro 5-4864. The concentrations of BZs needed to inhibit Ca2+ influx and TRH binding were in the micromolar range. These data show that BZs can modulate Ca2+ channel activity in endocrine cells and that these sites are distinct from those that modulate TRH binding on pituitary cells.