Ca2+ signaling was studied in pancreatic polypeptide (PP)-secreting cells isolated from mouse islets of Langerhans. After measuring the cytoplasmic Ca2+ concentration ([Ca2+]i), the cells were identified by immunocytochemistry. Most PP-cells reacted to carbachol and epinephrine with prompt and reversible elevation of [Ca2+]i, often manifested as slow oscillations. The carbachol effect was muscarinic, because it was inhibited by atropine. Beta-adrenergic elevation of cAMP explains the epinephrine stimulation, which was mimicked by an activator of adenylate cyclase and blocked by an inhibitor of protein kinase A. The responses to carbachol and epinephrine apparently involve depolarization with opening of voltage-dependent Ca2+ channels, because the effects were prevented by the Ca2+ channel antagonist methoxyverapamil and by diazoxide, which activates ATP-dependent K+ (K(ATP)) channels. Being equipped with K(ATP) channels, the PP-cells often responded to tolbutamide or high concentrations of glucose with elevation of [Ca2+]i. Somatostatin reversed the [Ca2+]i elevation obtained by carbachol, epinephrine, tolbutamide, and glucose. These preliminary studies support the idea that glucose has a direct stimulatory effect on the PP-cells, which can be masked by locally released somatostatin. Expressing both K(ATP) channels and voltage-dependent Ca2+ channels, the PP-cells share fundamental regulatory mechanisms with other types of islet cells.