The ADP-sensitive phosphoenzyme intermediate (E1P) of sarcoplasmic reticulum ATPase was formed using CaATP as a substrate and release of its bound calcium was investigated. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of E1P has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for Ca2+, whereas the remainder is presumably the nucleotide binding site. The calcium bound to the substrate site was readily replaced by other divalent cations or lanthanide ions. These ions induced a pronounced change in the rate of Ca2+ release from the transport sites on the same phosphoenzyme. In E1P, whose substrate site had been deprived of a metal, Ca2+ release from the transport sites was fast. It was similar to the Ca2+ release from the normal E1P, whose substrate site was occupied by either Ca2+ or Mg2+, in that it was accelerated by increasing pH or high ATP and inhibited markedly by the treatment of the enzyme with phospholipase C. Release of Ca2+ from E1P, whose substrate site had been rendered metal-free, increased the fluorescence intensity of the enzyme-bound 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, which decayed rapidly upon addition of Mg2+ plus K+. This result suggests that the active site of the resultant divalent cation-free phosphoenzyme has a conformation similar to that for the normal ADP-insensitive phosphoenzyme, E2P. These data suggest that the basic mechanism for Ca2+ release from the phosphoenzyme may be similar whether or not the phosphoenzyme has bound metal at its substrate site, although this metal affects the rate of Ca2+ release extensively.