Background: The objective was to compare signal transduction pathways exploited by glucose and cell swelling in stimulating insulin secretion. Methods: Isolated rat (Wistar) pancreatic islets were stimulated in vitro by 20 mmol/l glucose or 30% hypotonic medium (202 mOsm/kg) in various experimental conditions. Results: Glucose did not stimulate insulin release in calcium free medium. Cell swelling-induced insulin release in calcium free medium, even in the presence of the membrane permeable calcium chelator BAPTA/AM (10 µmol/l). Protein kinase C (PKC) inhibitor bisindolylmaleimide VIII (1 µmol/l) abolished the stimulation of insulin secretion by glucose but did not affect the swelling-induced insulin release. PKC activator phorbol 12-13-dibutyrate (1 µmol/l) stimulated insulin secretion in medium containing Ca²⁺ and did not potentiate insulin secretion stimulated by hypotonic extracellular fluid. Dilution of the medium (10-30%) had an additive effect on the glucose-induced insulin secretion. Noradrenaline (1 µmol/l) abolished glucose-induced insulin secretion but did not inhibit hypotonic stimulation either in presence or absence of Ca²⁺. Conclusion: Glucose- and swelling-induce insulin secretion through separate signal transduction pathways. Hyposmotic stimulation is independent from both the extracellular and intracellular Ca²⁺, does not involve PKC activation, and could not be inhibited by noradrenaline. These data indicate a novel signaling pathway for stimulation of insulin secretion exploited by cell swelling.