Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca(++) in the presence of ATP.(1) This ATP-dependent Ca(++) uptake by RBCMF appears to be the manifestation of an active Ca(++) transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca(++)-stimulated Mg(++) ATPase (Ca(++) ATPase) and Ca(++) uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50 degrees C abolished both Ca(++) ATPase and Ca(++) uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca(++) ATPase and Ca(++) uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca(++) ATPase or Ca(++) uptake. Both Ca(++) ATPase and Ca(++) uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca(++) uptake is intimately linked to Ca(++) ATPase.