In cardiac muscle, a membrane-associated Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) phosphorylates the Ca²⁺-pumping ATPase in addition to its previously characterized substrates, phospholamban and Ca²⁺-release channel (ryanodine receptor). The phosphorylated amino acid in the Ca²⁺-ATPase has been identified as serine. Posphorylation of the Ca²⁺-ATPase is rapid and is reversible by a membrane-associated protein phosphatase. Ca²⁺-ATPase purified from cardiac SR underwent phosphorylation by exogenous CaM kinase, and the phosphorylated enzyme displayed twofold greater catalytic activity without alteration in its Ca²⁺-sensitivity. The phosphorylation of the Ca²⁺-ATPase was found to be isoform-specific in that the cardiac and slow-twitch skeletal muscle isoform (SERCA2), but not the fast- twitch skeletal muscle isoform (SERCA 1), underwent phosphorylation by CaM kinase. Studies using SERCA 1 and SERCA 2 isoforms and their mutants expressed in a heterelogous cell system have resulted in i) confirmation of the isoform specificity of Ca²⁺-ATPase phosphorylation by CaM kinase, ii) identification of Ser³⁸ as the site in SERCA 2 phosphorylated by CaM kinase, and iii) demonstration of phosphorylation-induced increase in Vmax of Ca²⁺ transport by the SERCA 2 enzyme. These observations suggest that in cardiac and slow-twitch skeletal muscle direct phosphorylation of the SR Ca²⁺-ATPase by the membrane-bound CaM kinase may serve to stimulate Ca²⁺ sequestration and therefore, the speed of muscle relaxation.