Acanthamoeba myosin I heavy chain kinase activates the actin-activated Mg2+ -ATPase activity of the Acanthamoeba myosin I isoenzymes, myosins IA and IB, by phosphorylating a single site within the myosin heavy chain. In this paper, we report that myosin I heavy chain kinase also phosphorylates isolated turkey gizzard smooth muscle myosin light chains, gizzard smooth muscle heavy meromyosin, and intact gizzard smooth muscle myosin, all in the absence of Ca2+ and with specific activities close to those measured for purified Ca2+/calmodulin-dependent gizzard smooth muscle myosin light chain kinase. Myosin I heavy chain kinase incorporates a maximum of 2 mol of phosphate/mol of heavy meromyosin, both by itself and together with smooth muscle myosin light chain kinase (the light chain kinase alone incorporates 1.6 mol of phosphate/mol of heavy meromyosin). Both kinases phosphorylate intact smooth muscle myosin to a maximum of 2 mol of phosphate/mol of myosin. Myosin I heavy chain kinase fully activates the actin-activated Mg2+ -ATPase of both myosin and heavy meromyosin. Two-dimensional tryptic peptide maps of isolated light chains phosphorylated by myosin I kinase show the same phosphopeptide as for light chains phosphorylated by the light chain kinase. These results support the conclusion that myosin I heavy chain kinase phosphorylates gizzard smooth muscle myosin at the same site within the 20,000-Da light chain as does smooth muscle myosin light chain kinase. The results suggest that the amino acid sequence around the phosphorylation site within the heavy chain of Acanthamoeba myosin I isoenzymes may be similar to the primary sequence around the phosphorylation site within the smooth muscle myosin light chain.