The glutamic acid residue Glu 7 7 1 in the fifth transmembrane segment M5 of the Ca 2 + -ATPase of rabbit fast twitch muscle sarcoplasmic reticulum was substituted with lysine, alanine, and glycine by site-directed mutagenesis. Mutant Glu 7 7 1 ->Lys was unable to occlude Ca 2 + , and Ca 2 + did not inhibit phosphorylation from Pi or activate phosphorylation from ATP of this mutant. Mutants Glu 7 7 1 ->Ala and Glu 7 7 1 ->Gly were likewise unable to occlude Ca 2 + , but Ca 2 + in the millimolar concentration range activated phosphorylation from ATP and inhibited phosphorylation from P i of these mutants. The dephosphorylation of the ADP-insensitive E2P phosphoenzyme intermediate of mutants Glu 7 7 1 ->Ala and Glu 7 7 1 ->Gly was found to be blocked, whereas the dephosphorylation proceeded rapidly for mutant Glu 7 7 1 ->Lys. This finding suggests a role of the positive charge of the lysine in induction of dephosphorylation, supporting the hypothesis that the side chain of Glu 7 7 1 participates in the countertransport of two protons per Ca 2 + -ATPase cycle.