Purified cytochrome a was prepared from heart muscle by extraction with 0.1 [image] phosphate buffer (pH 7.4), resolution with cholate, fractionation by ammonium sulfate between 0.35 and 0.55 saturation, precipitation of contaminating cytochrome b and c1 at pH 7.4 in presence of 0.5% cholate and 0.25 saturation of ammonium sulfate, refractionation with ammonium sulfate between 0.25 and 0.35 saturation, dialysis against 0.02 [image] Na2HPO4 solution, adsorption on alumina Cgamma gel, elution with 0.07 [image] Na2HPO4 solution containing 0.5% cholate, and precipitation with ammonium sulfate at 0.35% saturation. This cytochrome a preparation shows the following properties: absorption bands at 280, 424, 600 m[mu] , (the oxidized form) and 444, 605 m[mu] (the reduced form); Fe 0.063, Cu 0.28 micromoles/mg N; 20% HC1 in acetone yields a dichromatic heme (585, and 430 m[mu] as pyridine hemochromogen); the preparation is contaminated by about 30% impurities (protein) when determined by ultracentrifugation and electrophoresis; no proportional relationship between activity and the Cu content; reduced by Na2S2O4, p-phenyl-enediamine, reduced cytochrome c, but not by hydroquinone or ascorbic acid; reoxidized by K3Fe(CN)6 or O2.