Information on the amino acid sequences of the internal peptide fragments of cytochrome b₅ from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase Sal1-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b₅. On the basis of nucleotide sequences of both cytochrome b₅ genomic gene and cDNA, the genomic cytochrome b₅ gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine→guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b₅ showed significant similarities with those of cytochrome b₅s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b₅ gene was expressed to 16% of the total soluble protein in Eseherichia coli. The holo-cytochrome b₅ accounted for 8% of the total cytochrome b₅ in the transformants. The purified cytochrome b₅ showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b₅.