Coproporphyrinogen oxidase has been located in the cytosol of yeast cells. The enzyme was purified to homogeneity from a heme mutant strain exhibiting a high specific activity (15–20 enzyme units/mg soluble protein compared to 1–2 enzyme units/mg soluble protein of the wild-type strain). The final preparation was homogenous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Mr= 35000) and isoelectrofocusing (pI=6.2). Gel filtration on AcA 44 gave a relative molecular mass of 70 000. N-terminal amino-acid sequence analysis revealed a single polypeptide chain. Thus the enzyme appears to be a dimer with identical subunits. Two iron atoms/molecule of native protein were detected; they could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. However the involvement of the iron atoms in the oxidative catalytic activity of the enzyme was not demonstrated. The Km value for coproporphyrinogen was 0.05 μM. The enzyme was active only when molecular oxygen was used as electron acceptor; no anaerobic activity could be detected. Thiol-directed reagents partially inhibited the enzyme, indicating that an SH group is required for activity. Yeast coproporphyrinogen oxidase was activated by phospholipids or neutral detergents as described for the bovine liver enzyme.