Three cDNA clones, designated 4f-8, 4f-34 and 4f-41, coding for three new forms of cytochrome P450 belonging to the CYP4F subfamily were isolated from an untreated rat brain cDNA library. cDNA 4f-8, 4f-34 and 4f-41 coded for proteins of 522, 526 and 537 amino acids, respectively, and their amino acid sequence similarity to CYP4F1 ranged from 71 to 80%. These new P450s were thus named CYP4F4, 4F5 and 4F6, respectively. Northern blot analysis revealed that the expression levels of these forms of P450 in the brain were somewhat low and that similar forms of subfamily 4F P450 were expressed in liver and kidney at a relatively high level compared with brain. No CYP4A expression was detected by Northern blot analysis in untreated rat brain mRNA. All three clones were in vitro-translatable using a reticulocyte lysate system. These results show that multiple forms of subfamily 4F P450 exist in the brain and that the subfamily 4F P450 may be one of the major forms of P450 in the brain.