A phosphorylation occurs at two sites in chicken gizzard myosin light chain kinase that appears to be catalyzed by an autophosphorylation reaction. This reaction is inhibited by approximately 75% in the presence of Ca2+-calmodulin, but is unaffected by the cAMP-dependent protein kinase inhibitor. Whereas the catalytic subunit of cAMP-dependent protein kinase phosphorylates myosin light chain kinase at only serine residues, the non cAMP-dependent phosphorylation occurs at both serine and threonine residues. One, if not both, of these latter sites are distinct from the sites recognized by the catalytic subunit of cAMP-dependent protein kinase. Consequently, there must be at least three and possibly four sites in myosin light chain kinase capable of incorporating phosphate, either in response to catalytic subunit or by autophosphorylation.