Cytochrome aa3 was purified 35- to 40-fold from submitochondrial particles of commercial bakers' yeast. The purification procedure involved solubilization of the enzyme with cholate, fractionation with ammonium sulfate, and chromatography on DEAE-cellulose in the presence of Triton X-100. The purified, active enzyme contained approximately 10 nmoles of heme a per mg of protein and was free of other hemoproteins. Upon sucrose gradient centrifugation in the presence of Triton X-100, it sedimented as a single peak with an apparent molecular weight of 190,000 to 225,000. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate resolved the enzyme into six major polypeptide bands of apparent molecular weight 42,000, 34,500, 23,000, 14,000, 12,500, and 9,500. The six components could be specifically precipitated from a crude mitochondrial extract with rabbit antiserum against the holoenzyme. The six components behaved as a single species during DEAE-cellulose chromatography and sucrose gradient centrifugation, and all could be precipitated with an antiserum against the three small components only. These and other observations indicate that all six components are physically associated with cytochrome c oxidase, even after solubilization of the mitochondrial inner membrane.