The reaction of N-ethylmaleimide (NEM) with three different forms of beef heart cytochrome oxidase was studied. Whereas denatured oxidase binds 4–5 mol NEM per heme a, native oxidase and a new form of the oxidase prepared by extensive dialysis against water each binds 0.8–1.0 mol NEM per heme a. Oxidase in the form of coupled vesicles binds only half as much NEM as the native molecule. Six different polypeptides are associated with cytochrome oxidase. They have molecular weights of (A) 45 000; (B) 25 000; (C) 16 000; (D) 12 500; (E) 10 500; and (F) 8500. Four of the six different subunits appear to contain groups that bind NEM. In the native oxidase subunits A, B, D, and F react with NEM, subunit B being the most reactive, subunit F the least. Water-dialyzed oxidase binds NEM to subunits A, B, and F, the binding to D being greatly diminished. Vesicular oxidase binds NEM to only subunit B.