The rat liver cDNA library in lambda gt11 was screened with the antisera against rat liver lanosterol 14 alpha-demethylase (P450(14DM)), and one clone (pRT-9) was isolated. The open reading frame of pRT-9 cDNA showed high homology to yeast P450(14DM) (48.9% in nucleotides and 37.7% in amino acids). Homology of the heme-binding region of pRT-9 protein to that of yeast P450(14DM) was 80%. Three additional regions showing more than 60% homology were identified and one of them was assignable as the distal helix. These findings suggest that pRT-9 protein is P450(14DM) and P450(14DMs) of rat and yeast may be orthologous. This is the first evidence suggesting the occurrence of orthologous P450 in widely diverged species.