Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.