Abstract: The expression of mitochondrial P450 in yeast is disclosed. The mitochondrial P450 includes a chimeric P450 in which a signal sequence present at the N-terminus of a mammalian mitochondrial P450 has been substituted by a signal of a yeast mitochondrial protein and a chimeric P450 in which the latter signal has been further substituted by a targeting signal sequence to microsome.An expression plasmid for producing the enzyme in a large scale, a recombinant yeast strain carrying the expression plasmid, a process for producing the enzyme by the use of the recombinant yeast, and a process for producing 5.beta.-cholestane-3.alpha., 7.alpha., 12.alpha., 27-tetrol, 25-hydroxyvitamin D.sub.3 and 1.alpha.,25-dihydroxyvitamin D.sub.3 are also disclosed.