In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr(166) by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an similar to 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr(289) and that, subsequent to Thr(166) phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr(289) is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283 - 288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr(166) phosphorylation. Simple removal of this region resulted in a significant increase in the k(cat) of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr(166) phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.