We examined the effect of hyperosmolality on exocytotic catecholamine secretion in isolated-perfused bovine adrenals, in both the absence and presence of Ca2+ in the extracellular medium. Exposure of adrenal glands to hyperosmotic media (600 mOsm/Kg H2O) for 20 minutes significantly decreased spontaneous catecholamine output, and was independent of extracellular Ca2+ concentration. Adrenal glands were exposed to a hyperosmotic Locke solution (600 mOsm/Kg H2O) for 10 minutes and then stimulated with acetylcholine. Hyperosmolality produced a significant, reversible inhibition of acetylcholine-evoked catecholamine secretion. As the osmotic strength of the perfusion fluid was increased, either by addition of sucrose or NaCl, there was a progressive reduction in the acetylcholine response. Hyperosmolality also decreased catecholamine release induced by 56 mM K+ and by removal of NaCl from the perfusion medium. Similar results were seen in glands exposed to a NaCl-free solution in the absence of extracellular Ca2+. These results suggest that inhibitory effects of hyperosmolality on stimulus-secretion coupling in the adrenal medulla are exerted after Ca2+ enters the chromaffin cells, and support one of the primary criteria of the chemiosmotic hypothesis.