Structural change of fragmented sarcoplasmic reticulum (SR), which is closely related to Ca2+-Mg2+-dependent ATPase [ATP phosphohydrolase, EC 3. 6. 1. 3] reaction, was investigated using spin label technique. SR was incubated with N-(l-oxyl-2, 2, 6, 6-tetramethyl-4-piperidinyl)maleimide under various conditions. The ESR spectrum, the Ca2+-Mg2+-dependent ATPase activity and the formation of phosphoryl intermediate, of the labelled SR, were measured, and the following results were obtained. 1. When SR was labelled with spin label reagent at pH 8.5 in the presence of ATP, alteration of ESR spectrum of labelled SR was produced on addition of various con-centrations of ATP (20μM-l mM) in the presence of Mg2+ (1 mM) and Ca2+ (0.1 mM). To maintain ATP concentration to a constant level, phosphoenol pyruvate-pyruvate kinase [EC 2. 7. 1. 40] system was used as an ATP-generating sourse. 2. The labelled SR was preincubated with EGTA or EDTA prior to adding ATP to chelate free Ca2+ and Mg2+. On adding ATP, the ESR spectrum of the labelled SR was not altered in the presence of these chelating reagents. The ATP-induced change in ESR spectrum, which had been inhibited by the existence of 0.5 mM EGTA, was restored by the subsequent addition of l mM CaCl2, in the presence of Mg2+. 3. Ca2+-Mg2+-dependent ATPase activities of the SR, which was labelled at pH8.5 in the presence of ATP and in its absence, were reduced to about 60% and to about 50% respectively of those of the control SR. However, incubation of SR with the spin label reagent did not appreciably reduced the amount of the phosphorylated intermediate, E∼32P, formed 5 sec after adding γ-32P-ATP to the SR. Furthermore, the existence of ATP during the incubation of SR with the spin label reagent did not virtually affect the amount of E∼32P formed 5 sec after adding γ-32P-ATP to the labelled SR. 4. On the basis of these and other results, the conformational change in SR induced by ATP dependent upon Ca2+ and Mg2+ was discussed in relation to the formation and decomposition of phosphoryl intermediate in the ATPase reaction and the Ca2+- translocation.