We reported that human GH (hGH) increased [Ca²⁺]_i and proliferation in pancreatic -cells (Sjoholm et al, J Biol Chem, 2000, 275, 21033) and that the hGH-induced rise in [Ca²⁺]_i involved Ca²⁺-induced Ca²⁺ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang et al, Mol Endocrinol, 2004, 18, 1658). We have now investigated the tyrosine kinases that convey hGH-induced rise in [Ca²⁺]_i and insulin exocytosis in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of JAK2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. While the hGH-stimulated rises in [Ca²⁺]_i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor-II, the inhibitors had no effect on insulin secretion stimulated by a high concentration of K⁺. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analogue PP3, suppressed the [Ca²⁺]_i elevation, and completely abolished insulin secretion stimulated by hGH but did not affect the responses to K⁺. Ovine prolactin increased [Ca²⁺]_i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca²⁺]_i, but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca²⁺]_i. Our study indicates that hGH stimulates rise in [Ca²⁺]_i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells.