The phosphorylation state of myosin in intact platelets has been investigated with alkaline urea-polyacrylamide gel electrophoresis. In gels of control cells, a band was found that co-migrated with the dephosphorylated form of isolated platelet myosin 20,000-dalton light chain. Stimulation of the cells by thrombin produced a dose-dependent shift of this band to the same position as that of the phosphorylated light chain. Conversion to the phosphorylated position was both complete and saturable with respect to thrombin concentration. Two-dimensional polyacrylamide gel electrophoresis was used to confirm the identity of this band as the 20,000-dalton myosin light chain. When (32P)PO4-labeled platelets were used, a direct correlation was found between the position of the light chain on the alkaline urea gel and the radioactivity. Our results demonstrate that in resting platelets myosin exists mainly in the dephosphorylated state and that stimulation by thrombin can produce a shift to the totally phosphorylated state.