Summary Cytochrome oxidase, a typical membrane enzyme, was purified by a simple procedure using immobilized cytochrome c. Sepharose-bound cytochrome c packed in a column was reduced with ascorbate. A cytochrome oxidase sample prepared from mitochondrial inner membrane was charged onto the column. Proteins containing no heme a were eluted from the column in the void volume. The oxidase was then eluted with increasing concentration of deoxycholate. The purified oxidase contains 13.6 nmoles heme a per mg protein, i.e. has a minimum mass of 73,500 daltons per heme a.