Protein kinase C was purified to the apparent homogeneity from a 100, 000×g supernatant fluid of human platelets homogenate, employing the steps of DEAE cellulose, Ultrogel AcA-34 and phenyl-Sepharose CL-4B chromatographies. The purified enzyme appeared as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, having a Mr 82, 000. With histone H-1 as a substrate its activity was 20-fold higher in the presence of phospholipid and Ca²⁺ than in the presence of phospholipid and EGTA, EGTA or Ca²⁺ alone. The enzyme has a K_m of 8.3μM and a V_max of 1.5μmol/mg/min for histone H-1. Platelet protein kinase C undergoes an apparent autophosphorylation. The enzyme was most active at 10-20mM mg²⁺ with histone H-1 as a substrate, wherease with mixed myosin light chains from turkey gizzard the maximum activity was observed at 1mM mg²⁺. Of several myosin light chains isolated from a variety of sources, the enzyme phosphorylates the 20, 000-dalton light chain of myosin from human platelets and turkey gizzard smooth muscle at a significant rate. The enzyme also phosphorylates the two phosphorylatable light chains of myosin from Limulus. These results suggest that platelet protein kinase C can phosphorylate the regulatory light chain of myosin non-muscle and smooth muscle, in which myosin phosphorylation catalyzed by myosin light chain kinase is thought to control contraction and cytoskeletal related processes.