Cell swelling causes an immediate secretory response in various cell types. This response shares similar dynamics with other types regulated secretory pathway but expresses some unique features (Ca2+ independence) leading to hypothesis that it utilizes novel pathway(s). This study was undertaken to investigate the functional role of intracellular calcium, PKC, PLA2, G-protein and chloride channels in hypotonic medium-induced insulin secretion. In vitro experiments were made by static incubations of isolated islets from Wistar rats and insulin release was measured by RIA. In contrast to glucose, osmotically stimulated secretion of insulin from pancreatic islets was not inhibited by bisindolyl maleimide VIII a PKC inhibitor in presence or absence of Ca2+ in medium. Inhibition of PLA2 by bromoenol lactone was also ineffective. Similarly inhibition of G-proteins by mycophenolic acid did not affect hypotonicity-induced insulin secretion. Inhibition of chloride channels with DIDS or acyclovir did not inhibit insulin secretion. Conclusion: Cell swelling induced insulin secretion utilizes unique novel signaling pathway. Supported by project 23191/23 of the Grant Agency VEGA, APVT-51-016002 and SP 51/0280800/0280802.