A new form of P450 designated P450ib2 was purified from rabbit small intestine microsomes. This P450 had properties very similar, to P450ib (CYP2J1), and showed 88% identity with CYP2J1 in its first 20 NH2-terminal amino acid sequence, excluding 3 undetermined residues. Both P450ib and P450ib2 were immunohistochemically detected in the mucosal epitherial cells of the duodenum, jejunum, and ileum in the small intestine, whereas no immunoreactivity was observed in other tissues including liver, kidney, lung, colon, and stomach. The results support that the two closely related P450s are specifically localized in the rabbit small intestine. Another small intestinal P450, P450ia, was found to hydroxylate a wide variety of fatty acids including straight-chain, branched-chain, unsaturated, or hydroxy fatty acids, and prostaglandin A at the omega and (omega-1) positions. P450ia was identical with a rabbit kidney fatty acid omega-hydroxylase, CYP4A7, in its 25 NH2-terminal amino acid sequence, excluding 2 undetermined residues. The results identify P450ia as CYP4A7.