A 7.0 kb DNA fragment was cloned by reverse genetics, which was located in the gene cluster of nikkomycin biosynthesis of Streptomyces ansochromogenes. Besides sanF--a gene related to nikkomycin biosynthesis of S. ansochromogenes, the sequence of a 2.2 kb BglII DNA fragment upstream sanF was determined. This 2.2 kb DNA fragment cointains two complete open reading frames (ORF). ORF1 consists of 1233 base pairs, ORF2 consistis of 195 base pairs, and they encode proteins with 410 amino acids and 64 amino acids respectively, which were designated as sanH and sanI. In search of databases, the deduced products of sanH has 46% amino acid identities and 62% amino acid similarities in comparison with the cytochrome P450 of Streptomyces griseolus, and the deduced protein of sanI has 56% amino acid identities and 70% amino acid similarities with the ferredoxin of S. griseolus. The function of sanH gene was studied using strategy of gene disruption, and the result showed that the disruption of sanH didn't influence nikkomycin biological activity in S. ansochromogenes.