The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S.Z., Xie, X. S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase-and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3-, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.