We examined the roles of myosin light-chain kinase in platelet responses to ADP using wortmannin, which almost completely inhibited myosin light-chain kinase at 3-6 microM. This concentration of wortmannin did not affect ADP-induced changes in the shape of the platelets, but it markedly inhibited aggregation in platelet-rich plasma and washed platelets. ML-9, another inhibitor of myosin light chain kinase, elicited similar effects on the platelet responses to wortmannin. Electron microscopic studies showed that there was no wortmannin effect on the ADP-induced spheration of discoid platelets, pseudopod formation, or granule centralization. Wortmannin at concentrations which prevented myosin light-chain kinase also inhibited platelet aggregation induced by ADP in the presence of U46619, an analogue of thromboxane A2, which is a prerequisite for ADP-induced irreversible aggregation. Although wortmannin partially inhibited protein kinase C, the protein kinase C inhibitor Ro-31-7549 (5 microM) prevented neither ADP- or ADP/U46619-induced changes in the shape of the platelets nor aggregation. These results suggest that myosin light-chain kinase activation is a prerequisite for ADP-induced platelet aggregation, but not for changes in their shape.