This study has explored the role of the pneumococcal toxin, pneumolysin (Ply), in activating human platelets. Following exposure to Ply (10-80 ng/ml), platelet activation and cytosolic Ca(2+) concentrations were measured flow cytometrically according to the level of expression of CD62P (P-selectin) and spectrofluorimetrically, respectively. Exposure to Ply resulted in marked upregulation of expression of platelet CD62P, achieving statistical significance at concentrations of 40 ng/ml and higher (P < 0.05), in the setting of increased influx of Ca(2+). These potentially pro-thrombotic actions of Ply were attenuated by depletion of Ca(2+) from the extracellular medium or by exposure of the cells to a pneumolysoid devoid of pore-forming activity. These findings are consistent with a mechanism of Ply-mediated platelet activation involving sub-lytic pore formation, Ca(2+) influx, and mobilization of CD62P-expressing α-granules, which, if operative in vivo, may contribute to the pathogenesis of associated acute lung and myocardial injury during invasive pneumococcal disease.