ATPase activities for the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted into dioleoylphosphatidylethanolamine [di(C18:1)PE] are, at temperatures higher than 20 degrees C, lower than in dioleoylphosphatidylcholine [di(C18:1)PC], whereas in egg yolk phosphatidylethanolamine the activities are the same as in di(C18:1)PC up to 25 degrees C, suggesting that low ATPase activities occur when the phosphatidylethanol-amine species is in the hexagonal H11 phase. ATPase activities measured in mixtures of di(C18:1)PC and di(C18:1)PE do not change with changing di(C18:1)PE content up to 80%. It is concluded that curvature frustration in bilayers containing di(C18:1)PE has no effect on ATPase activity. The rates of phosphorylation and of Ca2+ transport are identical for the native ATPase and for the ATPase in di(C18:1)PE. Dephosphorylation of the phosphorylated ATPase in di(C18:1)PE at 25 degrees C is, however, slower than for the native ATPase, explaining the lower steady-state rate of ATP hydrolysis; in egg yolk phosphatidylethanolamine at 25 degrees C the rate of dephosphorylation is equal to that for the unreconstituted ATPase. Phosphorylation of the ATPase by P1 in the absence of Ca2+ is unaffected by reconstitution in di(C18:1)RE. The stoichiometry of Ca2+ binding to the ATPase is also unaltered. Studies of the effect of di(C18:1)PE on the fluorescence intensity of the ATPase labelled with 7-chloro-4-nitro-2,1,3-benzoxadiazole are consistent with an increase in the E1/E2 equilibrium constant, where E1 is the conformation of the ATPase with two high-affinity binding sites for Ca2+ exposed to the cytoplasm, and E2 is a conformation unable to bind cytoplasmic Ca2+. A slight increase in affinity for Ca2+ can be attributed to the observed increase in the E1/E2 equilibrium constant.