Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol.h-1.mg-1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic-acid-precipitated total-liver-protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 microM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.