We investigated the mechanism underlying somatostatin-induced increase in [Ca2+]i leading to insulin release in the presence of arginine vasopressin (AVP) in clonal beta-cell HIT-T15. Somatostatin increased [Ca2+]i and insulin release in a biphasic pattern, characterized by a sharp and transient increase followed by a rapid decline to the sub-basal level. Pretreatment with pertussis toxin, which inactivates Gi/Go, abolished the effects of somatostatin. U-73122, an inhibitor of phospholipase C, antagonized somatostatin-induced increase in [Ca2+] i. In Ca2+-free environment, somatostatin still increased [Ca2+]i, whereas depletion of intracellular Ca 2+ stores with thapsigargin, a microsomal Ca2+ ATPase inhibitor, abolished somatostatin's effect. In the presence of bradykinin, another Gq-coupled receptor agonist, somatostatin also increased [Ca 2+]i, but not in the presence of isoproterenol (a Gs-coupled receptor agonist) or medetomidine (a Gi/Go-coupled receptor agonist). Utilizing selective agonists for each somatostatin receptor subtype (SSTRI-5) and PRL-2903, a specific SSTR2 antagonist, we characterized the receptor mediating the somatostatin signaling. In the presence of AVP, treatment with the SSTR2 agonist L-779,976 resulted in responses similar to those seen with somatostatin. L-779,976 increased both [Ca2+]i and insulin release in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca2+] i or basal insulin release. In the presence of AVP, all other somatostatin receptor agonists failed to increase [Ca2+]i and insulin release. The effects of somatostatin and L779,976 were abolished by PRL-2903. Administration of antibody against the beta subunit of Gi/Go into single cells inhibited the increase in [Ca2+]i by somatostatin, but antibodies against Gialpha1/Gialpha2 and Gialpha3/Goalpha failed to do so. Somatostatin increased PIP2 synthesis from PIP in the presence and absence of AVP, whereas an increase in IP3 synthesis was observed only in the presence of AVP. Taken together, our study strongly suggests that activation of the SSTR2 coupled to Gi/Go by somatostatin increases PIP2 synthesis through the betagamma dimer. The PIP2 generated by somatostatin serves as additional substrate for preactivated PLC-beta, which hydrolyzes PIP2 to form IP3, leading to Ca2+ release from the endoplasmic reticulum and insulin release in clonal beta-cell HIT-T15. The increases in [C2+]i and insulin release are due to a cross-talk between Gq and Gi/Go, although not limited to the AVP and somatostatin receptors.