We report the isolation and purification of synhibin, a new M r 68000 protein, which inhibits synexin. Synexin mediates Ca 2 + -dependent chromaffin granule aggregation and fusion, processes perhaps important during exocytosis. Our data indicate that synhibin action involves competition with synexin for a site on the chromaffin granule membrane involved in membrane contact. Synhibin may thus be an important intracellular regulator of synexin action during secretion.