The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca 2 + binds only to E and interior Ca 2 + binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of c E C a 2 , the enzyme with Ca 2 + bound at the exterior site, was measured at different Ca 2 + concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca 2 + by reaction with 0.5 mM [γ- 3 2 P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO 4 , 40 mM Mops/KOH, pH 7.0, 25 o C). The sigmoidal dependence on free exterior calcium concentration of the concentration of c E C a 2 , measured as [ 3 2 P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K 0 . 5 is 2.8 μM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca 2 + to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca 2 + is determined by the state of phosphorylation of the CaATPase.