Gene deletion vector pXL05(pKC1139∷△olmA1 +△olmA4) was used to disrupt oligomycin PKS encoding genes (olmA) in Streptomyces avermitilis CZ8-73, the producer of anthelmintic avermectins B and the cell growth inhibitor oligomycin. olmA gene cluster in the chromosome was displaced by deletion allele on the plasmid via double crossover. Four of disruptants were confirmed by Southern blotting. Shaking flask experiments and HPLC analyses showed that the four mutants no longer produced the toxic oligomycin, but only made four components of avermectins B, which were avermectin B1a, B1b, B2a, B2b. The yields of avermectins B in these mutants were separately equal to those in CZ8-73. This revealed that olmA genes deletion did not affect the biosynthesis of avermectins. The deletion mutants were proved to be genetically stable, and thus might be promising strains in industrial production of avermectins B.