The role of chemiosmotic lysis in the exocytotic release of insulin has been studied using perifused rat pancreatic islets of Langerhans. Established criteria for osmotic lysis of secretory granules requires proton translocation across the secretory granule membrane and the influx of a permeant anion. The consequent increase in granule osmolarity induces water entry and granule lysis. A proton gradient has been previously established to exist across the insulin secretory granule membrane. We have examined the sensitivity of insulin release to 1) hyperosmolar solutions, 2) replacement of medium Cl-, 3) replacement of medium Na+, and 4) anion transport inhibitors. The addition of 200-600 mM sucrose resulted in a 32-69% inhibition of insulin release due to 16.7 mM glucose. Replacement of Cl- by isethionate or SO4--reversibly inhibited glucose-induced insulin release by 47% and 78%, respectively. Na+ replacement by choline did not influence the secretory response. 4,4'-Diisothiocyano-2,2'-stilbene disulfonic acid (500 microM) and probenecid (10 mM) inhibited insulin release by 73% and 79%, respectively. These drugs are known to inhibit anion exchange in erythrocytes and may be influencing Cl- entry into the secretory granule fused to the plasma membrane by a similar mechanism. Furosemide inhibits NaKCl2 cotransport in erythrocytes, but had no influence on glucose-induced insulin release, suggesting that Cl- does not enter the secretory granule by this pathway. The primary criteria for the participation of a chemiosmotic mechanism subserving lysis of the insulin secretory granule are fulfilled by these results.