Quenched-flow mixing was used to characterize the kinetic behavior of the intermediate reactions of the skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase (SERCA1) at 2 and 21 °C. At 2 °C, phosphorylation of SR Ca-ATPase with 100 μM ATP labeled one-half of the catalytic sites with a biphasic time dependence [Mahaney, J. E., Froehlich, J. P., and Thomas, D. D. (1995) Biochemistry 34, 4864-4879]. Chasing the phosphoenzyme (EP) with 1.66 mM ADP 10 ms after the start of phosphorylation revealed mostly ADP-insensitive E2P (95% of EP total), consistent with its rapid formation from ADP-sensitive E1P. The consecutive relationship of the phosphorylated intermediates predicts a decrease in the proportion of E1P ([E1P]/[EP total]) with increasing phosphorylation time. Instead, after 10 ms the proportion of E1P increased and that of E2P decreased until they reached a constant 1:1 stoichiometry ([E1P]:[E2P] ∼ 1). At 21 °C, phosphorylation displayed a transient overshoot associated with an inorganic phosphate (P i) burst, reflecting increased turnover of E2P at the higher temperature. The P i burst exceeded the decay of the EP overshoot, suggesting that rephosphorylation of the enzyme occurs before the recycling step (E2 → E1). This behavior and the reversed order of accumulation of phosphorylated intermediates at 2 °C are not compatible with the conventional linear consecutive reaction mechanism: E1 + ATP → E1·ATP → E1P + ADP → E2P → E2·P i → E1 + P i. Solubilization of the Ca-ATPase into monomers using the nonionic detergent C 12E 8 gave a pattern of phosphorylation in which E1P and E2P behave like consecutive intermediates. Kinetic modeling of the C 12E 8-solubilized SR Ca-ATPase showed that it behaves according to the conventional Ca-ATPase reaction mechanism, consistent with monomeric catalytic function. We conclude that the nonconforming features of native SERCA1 arise from oligomeric protein conformational interactions that constrain the subunits to a staggered or out-of-phase mode of operation.