The reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30 degrees C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethyl[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activity occurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled peptides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.