The effect on cytosolic Ca2+ concentration ([Ca2+]i) of cAMP analogues and the adenylate cyclase-stimulating agents forskolin, isoproterenol and glucagon has been examined in an insulin-secreting beta-cell line (HIT T-15) using fura 2. All these manipulations of the cAMP messenger system promoted a rise in [Ca2+]i which was blocked by the Ca2+ channel antagonists verapamil and nifedipine or by removal of extracellular Ca2+. The action of the adenylate cyclase activator forskolin was glucose-dependent. The results suggest that cAMP elevates [Ca2+]i in HIT cells by promoting Ca2+ entry through voltage-sensitive Ca2+ channels, not through mobilization of stored Ca2+. Activation of Ca2+ influx may be an important component of the mechanisms by which cAMP potentiates fuel-induced insulin release.