Heme oxygenase (HO) isoforms in hematin and phenylhydrazine-induced Sprague-Dawley rat liver and brain were purified using DEAE-Sephacel and hydroxylapatite. Rat liver HO-1 and brain HO-2 were determined by the Western blotting analysis. The results showed that two isoforms were purified and identified from the induced rat liver, and the HO-1 was the predominant form with a ratio of 2∶1. In the native state, the activity of HO-2 was detected to be fully refractory to hematin and phenylhydrazine, whereas the activity of HO-1 was increased in response to these agents. The apparent molecular weights of HO-1 and HO-2 were about 30 ku and 36 ku respectively. In the untreated liver and treated brain, only one peak of HO activity (HO-2) was detected. The antiserum against liver HO-2 was employed in Western blotting analysis, cross-reactivity of HO-2 in the brain was observed, and that of HO-1 in the liver was not observed. The study suggests that HO-1 and HO-2 exist in the hematin and phenylhydrazine treated rat liver, HO-1 is an inducible enzyme, and only HO-2 exists in the treated rat brain. Two constitutive forms were different in molecular weight, inducibility and immunochemical properties.