Heme oxygenase was purified to apparent homogeneity from pig spleen microsomes. The purified heme oxygenase showed an apparent molecular weight of 157,000 +/- 7,000 daltons when estimated by gel filtration. On SDS-polyacrylamide gel electrophoresis, the heme oxygenase preparation gave a single protein band showing a minimum molecular weight of about 26,000 daltons. Heme oxygenase could readily bind with heme and the resulting heme complex gave an absorption maximum at 406 nm. The heme bound to the enzyme protein was found to be a good substrate for the heme oxygenase reaction.