The complex of Mg X ATP and the calcium adenosinetriphosphatase of sarcoplasmic reticulum (E X ATP) reacts with 50-300 microM Ca2+ to form phosphoenzyme (E-P X Ca2) with a rate constant of 70 s-1 (pH 7.0, 100 mM KCl, 5 mM MgSO4, 25 degrees C, and SR vesicles passively loaded with Ca2+). This rate constant is independent of Ca2+ concentration above 50 microM. It is 4-6 times faster than the rate constants of 11-15 s-1 for the conformational change associated with Ca2+ binding in the absence of activation by ATP. The reaction of 200 microM Ca2+ with enzyme preincubated in 0.9 microM [gamma-32P]ATP X Mg shows a burst of [32P]E-P X Ca2 formation. This result indicates that Mg X ATP bound to the active site, and not a regulatory site, can accelerate the conformational change associated with Ca2+ binding because this concentration of Mg X ATP is well below the Kd of 160-500 microM for the putative regulatory site. When an unlabeled ATP chase is added with the Ca2+ to enzyme preincubated with [gamma-32P]ATP X Mg, the amount of [32P]E-P X Ca2 that is formed increases with the concentration of ATP in the preincubation solution and is consistent with a maximum fraction trapped of 0.55 and Kd = 4.5 microM for the dissociation of Mg X ATP from the active site. The fact that labeled E X ATP can be trapped by added Ca2+ confirms the conclusion that dissociation of ATP from E X ATP X Ca2 is slow relative to phosphorylation.