Donald K. Blumenthal* andJames T. Stull abstract: Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was un-dertaken in order to determine the stoichiometries and equi-librium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to requirebinding of Ca2+ to all four divalent metal binding sites on calmodulin to form the complex, Ca42+* cal-modulin. This complex then interacts with the inactive cat-IVtyosin light chain kinase, the enzyme catalyzing the phosphorylation of the phosphorylatable or P light chain of myosin, was first identified in fast-twitch skeletal muscle (Pires et al., 1974). The enzyme was subsequently purified to ap-parent homogeneity and shown to be a monomer of approx-imately 80000 molecular weight (Pires & Perry, 1977; Ya-zawa & Yagi, 1978; Nairn & Perry, 1979). The purified enzyme specifically phosphorylated the P light chain of myosin, and was completely dependent on micromolar concentrations of Ca2+(Pires et al., 1974; Pires & Perry, 1977). It was later shown that the activity of myosin light chain kinase from smooth muscle (Dabrowska et al., 1977), cardiac muscle (Walsh et al., 1979), and skeletal muscle (Yazawa & Yagi, 1977) was also dependent upon nanomolar concentrations of a small molecular weight protein, subsequently identified as calmodulin1 (Barylko et al., 1978; Dabrowska et al., 1978; Yagi