The calcium-sensing receptor gene was recently shown to be expressed in rat pancreatic islets and purified islet B-cells. In this study, we investigated the possible role of this receptor in the regulation of insulin release from isolated rat pancreatic islets. Poly-l-arginine (0.2–0.3 μM) and poly-l-lysine (0.03–0.1 μM) increased insulin output evoked by d-glucose (8.3 mM). This positive effect faded out at higher concentrations of the basic peptides. Likewise, the release of insulin evoked by 8.3 mM d-glucose was significantly lower at high (1.0 mM) than low (0.05–0.1 mM) concentrations of neomycin. The insulinotropic action of Ba²⁺ in Ca²⁺-deprived islets was potentiated in rats pretreated with pertussis toxin. However, Gd³⁺ inhibited insulin release evoked by dd-glucose in islets prepared from normal rats or animals pretreated with pertussis toxin and incubated in the absence or presence of either theophylline or forskolin. Gd³⁺ (0.3 mM) failed to affect effluent radioactivity from islets prelabeled with myo-[2-³H]inositol and cyclic AMP net production in islets incubated in the absence or presence of forskolin. Gd³⁺ decreased, however, ⁴⁵Ca efflux from prelabeled islets perifused in the absence or presence of extracellular Ca²⁺. It is speculated that a negative insulinotropic action mediated by the calcium-sensing receptor, and possibly attributable to a fall in cytosolic Ca²⁺ concentration, may prevent excessive insulin secretion in pathological situations of hypercalcemia.