We have previously identified and partially purified a kinase from Acanthamoeba that catalyzes specifically the phosphorylation of the heavy chains of three related enzymes, Acanthamoeba myosins IA, IB and IC. Phosphorylation of their heavy chains had also been shown to be required for actin-activation of the Mg2 ion-ATPase activities of these three myosins. We have now shown a direct correlation between the extent of phosphorylation of the heavy chains of Acanthamoeba myosin IA and its actin-activated Mg2 ion-ATPase activity over a range of 0 to 0.8 moles phosphate per mole of heavy chain. We have now purified another kinase that specifically phosphorylates the heavy chain of a different myosin isoenzyme, Acanthamoeba myosin II. However, phosphorylation of the heavy chain, in this case, has no demonstrable effect on the actin-activated Mg2 ion-ATPase activity of the myosin. 32P is incorporated into the heavy chain of myosin II when cells are grown in the presence of (32P)phosphate and myosin II that is labelled in this way (1 mole 32P/mole heavy chain) does not incorporate additional phosphate when incubated with the kinase. thus, the phosphorylation of myosin II heavy chain occurs physiologically but its role is not known.