It is well known that phosphorylation of the membrane protein phospholamban by cAMP-dependent or Ca2+/calmodulin-dependent protein kinase results in the activation of the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR); such enzyme activation is thought to be due to the disruption of an inhibitory interaction of non-phosphorylated phospholamban with the ATPase. We describe here a novel mechanism for the regulation of the ATPase through direct phosphorylation of this enzyme by a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the SR membrane. It is shown that incubation of cardiac SR in the presence of Ca2+ and calmodulin results in the phosphorylation of the ATPase in addition to the previously recognized substrates of CaM kinase, viz. phospholamban and Ca2+ channel. The phosphorylated amino acid in the ATPase has been identified as serine. Phosphorylation of the membrane-bound ATPase is stimulated by exogenous CaM kinase. Furthermore, ATPase purified from cardiac SR is phosphorylated by exogenous CaM kinase and the phosphorylated enzyme displays 2-fold increase in catalytic activity without any appreciable change in its Ca2+ sensitivity. Thus, direct phosphorylation of the Ca(2+)-pumping ATPase by CaM kinase can stimulate its enzymatic activity and, therefore, Ca2+ transport function.