The total calcium content of secretory granules, Cag, was evaluated in isolated neurohypophysial nerve endings. The Cag in the resting state, as measured by X-ray microanalysis, is relatively high with an average of 7.4 +/- 0.6 mmol/kg wet weight. Following a depolarizing potassium challenge, a subpopulation of granules with even higher Cag could be detected, dispersed over a wider range of concentrations (up to 70 mmol/kg wet weight). After subsequent rinsing in physiological saline, Cag decreased to control values. This could have resulted from Ca2+ extrusion, or from preferential secretion of calcium-enriched granules. Our data can be interpreted in favor of the second explanation since no decrease in Cag was observed when secretion was blocked by a hyperosmotic saline. The effect of hyperosmotic conditions on isolated nerve endings was further studied by monitoring free cytoplasmic Ca2+ with the calcium-sensitive dye Fura-2 and by conventional electron microscopy. It was demonstrated that hyperosmotic treatment alone did not increase basal cytosolic Ca2+ concentrations but did significantly reduce the potassium-induced cytosolic rise in Ca2+. Electron microscopy of nerve endings in hyperosmotic conditions showed numerous exocytotic figures at various stages. The observed changes in Cag are in accord with a published hypothesis which proposes that intragranular calcium is a significant variable in regulated secretion.