The time-resolved kinetics of Ca2+ binding to the SR Ca-ATPase in the E1 state was investigated by Ca2+-concentration jump experiments. Ca2+ was released by an ultraviolet-light flash from caged calcium, and charge movements in the membrane domain of the ion pumps were detected by the fluorescent styryl dye 2BITC. The partial reaction (H3E1 ↔) E1↔CaE1↔Ca2E1 can be characterized by two time constants, τ1 and τ2, both of which are not significantly Ca2+-concentration-dependent and only weakly pH-dependent at pH<7.5. Both time constants differ by a factor of ∼50 (4.7 vs. 200ms). The weak substrate-dependence indicates that the rate-limiting process is not related to Ca2+ migration through the access channel and ion binding to the binding sites but to conformational rearrangements preceding the ion movements. The high activation energy obtained for both processes, 42.3kJ mol−1 and 60.3kJ mol−1 at pH 7.2, support this concept. Transient binding of Ca ions to the loop L67 and a movement of the Ca-loaded loop are discussed as a mechanism that facilitates the entrance of both Ca ions into the access channel to the ion-binding sites.