ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca 2+ and Mg > in the complete absence of the other cation. Increasing concentrations of Mg 2+ produced an apparent inhibition of the Ca>-dependent ATP hydrolysis. CDTA (trans-l,2-diaminocyclohexane-N,N,N' ,N'-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca 2+ with a high affinity (K0.~ Ca z+ = 0.41/,LM) suggesting the presence of a high affinity Ca2+-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca 2+ and Mg 2+ concentrations and failed to stimulate membrane phosphorylation and Ca 2+ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48/80 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity CaZ+-pumping ATPase previously characterized in dog heart and other species.