Myosin light chain kinase (MLCK) is a calmodulin-dependent protein kinase which phosphorylates the 20, 000 dalton regulatory light chain of myosin II. Here we show that activation of chicken gizzard MLCK by Ca²⁺/calmodulin is inhibited by autophosphorylation at 2 sites in the absence of Ca²⁺/calmodulin. Two phosphorylation sites are located in the functional domain of the kinase, the threonine site toward the actin binding domain near the N-terminus of MLCK and the serine site in immediate proximity to the calmodulin binding site. The autophosphorylation was significantly inhibited by the binding of calmodulin to MLCK in the presence of Ca²⁺.