To investigate the regulation of actin-myosin interaction in rabbit phagocytic cells, purified myosin and a partially purified cofactor protein were obtained from polymorphonuclear leukocytes (PMN) and alveolar macrophages (ALM) by molecular sieve filtration over an agarose column. ALM cofactor enhanced the Mg++-ATPase activity of ALM myosin by actin to 0.15 × 0.04 μmoles Piper mg myosin per min and PMN cofactor enhanced PMN myosin to 0.43 × 0.03 μmoles Piper mg myosin per min. The crude cofactor preparations isolated from the two types of leukocyte extracts were interchangeable with the leukocyte myosins. When ALM cofactor was added to a PMN actomyosin complex, the Mg++-ATPase activity of the PMN myosin was 3-fold higher than with ALM cofactor and its own actomyosin complex. In contrast, PMN cofactor did not enhance ALM actomyosin Mg++-ATPase activity beyond that observed with ALM cofactor and ALM actomyosin. Cofactor protein from the PMN was further purified on a DEAE-Sephagel column. After electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, the isolated fraction weighed 70,000 daltons. This fraction stimulated the actin-mediated myosin Mg++-ATPase. In the presence of Mg++and [γ32P]ATP, the 70,000 dalton protein phosphorylated the 20,000 dalton light chain of PMN myosin, leading to the incorporation of 0.62 × 0.09 moles of Piper mole myosin. On the basis of these results, we propose that phagocytic cofactor is a kinase which regulates the enzymatic activity of phagocytic cell myosin.