Platelet secretory granules were used for in vitro studies bearing on the mechanism of exocytosis. A morphologically homogeneous preparation of alpha-granules was obtained from porcine platelets. The granules were shown to be osmotically fragile, as determined by electron microscopy, turbidity measurements, and the release of protein upon hypotonic lysis. Lysis could also be induced in isotonic media by addition of gramicidin to granules suspended in NaCl or KCl. Increasing the osmotic strength of the medium prevented this effect. The antibiotic had no effect when added to granules in sucrose solution. Submillimolar concentrations of Ca2+ drastically reduced the turbidity of granule suspensions while releasing protein to the supernatant. Electron micrographs showed that Ca2+-treated granules lost most of their electron-dense core, indicating that lysis had occurred. Mg2+ and other divalent cations failed to induce this response. The effect occurred in NaCl or KCl but not in sucrose or sorbitol solutions. Increasing the osmotic content of the medium with sucrose suppressed the Ca2+-induced lysis. It is suggested that Ca2+ increases the ionic permeability of the granule membrane leading to solute influx followed by osmotically coupled water uptake and, therefore, swelling and lysis. During platelet activation, Ca2+-induced granule swelling could provide the driving force for membrane fusion leading to exocytosis.