Two cDNA clones designated 2d-29 and 2d-35 encoding a novel P450 belonging to the P450 2D subfamily were isolated from control and imipramine-treated rat brain libraries, respectively. cDNA 2d-29 and 2d-35 contained 1786 and 2754 bp nucleotides each. Although their 5'- and 3'-untranslated sequences were different from each other, their open reading frames were identical, encoding a protein which had 5 substitutions of amino acids in the C-terminal region compared with P450 2D4. Northern blot analysis showed that the expression levels of 2d-29 and 2d-35 in the brain were lower than that of P450 2D4 in the liver. However, it is noteworthy that the expression of 2d-35 was not detected in the liver but in the brain. These results suggest that a novel P450 is expressed in the brain under different regulation.