Rat liver microsomes have previously been shown to contain hemoproteins having molecular weights of 53,000, 50,000, and 45,000. The 45,000-dalton hemoprotein, which is induced in rat liver microsomes by pretreatment of animals with phenobarbital, is resistant to proteolysis by trypsin. This characteristic was used to purify it from the other microsomal hemoproteins. In the procedure used, a sodium cholate-solubilized microsomal fraction from phenobarbital-pretreated rats was treated with trypsin and chromatographed on Sephadex G-100 to separate the hemoprotein from preolytic degradation products. The hemoprotein thus isolated was homogenous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified spectrally as a cytochrome P-420 hemoprotein. This hemoprotein was free of cytochrome b5 and NADPH-cytochrome c reductase activity. Antibody prepared against the protease-treated cytochrome P-420 hemoprotein will not cross-react with the 53,000- and 50,000-dalton hemoproteins. This was assessed by three criteria. First, immunoprecipitation studies were conducted with detergent-solubilized partially purified cytochrome P-450 preparations isolated from the liver microsomes of control and phenobarbital- and 3-methylcholathrene-retreated rats. The antibody immunoprecipitated only the 45,000-dalton hemoprotein from these partially purified cytochrome P-450 preparations, each of which contains all three hemoproteins. Second, the antibody demonstrated specificity with regard to the microsomal hydroxylation reactions it would inhibit in a reconstituted hydroxylation system containing partially purified cytochrome P-450 (448) fractions isolated from the liver microsomes from phenobarbital- or 3-methylcholathrene-pretreated rats. The antibody would inhibit benzphetamine-N-demethylation catalyzed by both cytochrome P-450 fractions but would not inhibit benzpyrene hydroxylation catalyzed by either. Third, agglutination and complement fixation assays were performed to assess the binding of the antibody to liver microsomes isolated from control and phenobarbital- or 3- methylcholanthrene-pretreated rats. These studies demonstrated that the antibody binds preferentially to the liver microsomes isolated from phenobarbital-pretreated rats, in which the 45,000-dalton hemoprotein has been shown to be induced. It is hypothesized that there are very significant structural and catalytic differences among the cytochrome P-450 hemoproteins.