Escherichia coliwas used to express the two closely related cytochromes P450 2B1 and 2B2 and two mutants of 2B2 in which residues Gly-303 and Ala-363 were replaced by Ser and Val, respectively. The expressed proteins were partially purified and assayed for benzphetamine andn-octylamine (NOA) binding and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (EOD), benzphetamine N-demethylation (BND) and 7,12-dimethylbenzaanthracene (DMBA) hydroxylation activities in the presence and absence of cytochrome b5. TheKdvalues for benzphetamine and NOA obtained for the wild-type enzymes were similar to reported values. The Ala-363 → Val mutant (A363V) of 2B2 exhibitedKdvalues for both ligands that were more similar to 2B1 than to 2B2. The EOD and BND activities of the A363V mutant were 10- and 3.8-fold those exhibited by 2B2, respectively. With DMBA, the A363V mutation led to a 6-fold increase in the hydroxylation activity at the 7-methyl substituent while the hydroxylation activity at the 12-methyl substituent was slightly suppressed. The 7-hydroxymethyl:12-hydroxymethyl product ratio obtained with the A363V mutant (1.3) was much closer to the ratio obtained with 2B1 (1.9) than to that obtained with 2B2 (0.17). Conversely, the Gly-303 → Ser substitution did not influence the characteristics of the 2B2-catalyzed metabolism of DMBA to the same magnitude. When cumene hydroperoxide (CHP) was used to support the EOD activities of the proteins, 2B2 exhibited a 2- to 20-fold greater activity than 2B1 or either of the mutants. Examination of the CHP-derived products of the EOD reactions revealed the formation of mainly 2-phenyl-2-propanol due to the heterolytic cleavage of CHP. However, only the 2B1 EOD-reaction mixture also contained the P450-mediated CHP-isomerization products 2-phenyl-1,2-propanediol and 2-(p-hydroxyphenyl)-2-propanol. The formation of these products with 2B1 but not 2B2 may explain why 2B1 is not as efficient as 2B2 or 2B2-G303S in carrying out the CHP-supported reactions.