Micromolar levels of free calcium ions added to the extracellular medium elicit secretion of serotonin from electropermeabilized bovine platelets in the presence of millimolar levels of Mg-ATP. Such Ca²⁺-dependent secretion of serotonin was almost completely impaired when the permeabilized platelets were preincubated for 1 min at 35°C in 100 μM Ca²⁺ without Mg-ATP. The half-maximal effect was observed with about 45 μM Co in the preincubatlon medium. Inhibitors of serine-thiol protease, such as leupeptin and antipain, suppressed the impairment of the secretion of serotonin by the preincubation with Ca²⁺. Electron microscopic observation revealed that disorganization of the cytoskeletal structures, in particular of the membrane undercoat and the network of microfilainents, accompanied the impairment of secretion of serotonin. Microfilaments were also found to be dissociated from dense granules that contained serotonin. These morphological changes were also suppressed when antipain was included in the Ca²⁺-preincubation medium. Coincident with these morphological changes, the following biochemical changes were observed in 100 μM Ca²⁺ but not in the presence of Ca and antipain. The amount of Triton-insoluble cytoskeleton and the acto-myosin content of the dense-granule fraction were markedly decreased. The decrease in Triton-insoluble cytoskeletons was quantita tively correlated with the degree of Impairment of secretion of serotonin. Immunoblot analysis of EGTA extracts of the cells showed that the 240-kDa spectrin in platelets was degraded to a 235-kDa fragment, and a260-kDa actin-binding protein (ABP) in platelets was partially degraded to 190- and 11O-kDa components. A novel 88-kDa protein in the dense-granule fraction, which was shown to be an actin-binding protein by affinity chromatography on a DNase 1 column, was also found to be cleaved upon preincubation with Ca²⁺ in the absence of antipain. These results indicate that the Ca²⁺ secretion of serotonin is impaired, and that the cytoskeletal structures are at the same time seriously damaged through the activation of an endogenous protease activated by Ca Three actin-binding proteins, namely, spectrin, ABP, and an 88-kfla protein, were identified as substrates for the protease. They might play some positive roles in the secretion of serotonin from platelets.