We have recently shown [Mészáros, L. G., & Bak, J. (1992) Biochemistry 31, 1195-1200] that, during the rapid phase of Ca2+ uptake into sarcoplasmic reticulum (SR), internalization and binding of Ca2+ to the cytoplasmic high-affinity binding sites of the Ca2+ ATPase occur simultaneously, resulting in a transient supernumerary Ca/ATP stoichiometry. Here we address the question of whether the cytoplasmic high-affinity and the luminal low-affinity Ca2+ binding sites of the SR Ca2+ ATPase also coexist. SR vesicles were loaded with Ca2+ (0-10 mM), and then the kinetics of EP formation and decomposition as well as the maximum level of EP formed from radiolabeled ATP were determined at conditions which only allow single-cycle reactions to occur: empty or Ca-loaded SR vesicles (in micromolar extravesicular Ca2+) were either mixed with ATP plus millimolar EGTA or added in amounts that set a Ca2+ ATPase/ATP ratio of 80-85 at the initiation of the reaction. The rates of EP formation and decomposition were both significantly reduced in Ca-loaded, compared to empty (ionomycin-treated), vesicles. However, the value of EPmax was unaltered by Ca-loading, suggesting the existence of the enzyme intermediate, E.Ca2(cyt).Ca2(lum), i.e., the coexistence of the cytoplasmic and the luminal Ca2+ binding sites of the Ca-pump. These results suggest that the uphill transport of Ca2+ might not be based on an alternating relocation and conversion of the Ca2+ binding sites of the Ca2+ ATPase.