A new actinomycete strain, isolated from soil in China, strongly inhibited in vitro proliferation of human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines. The strain, designated L033, was identified as a strain of Streptomyces avermitilis based on cultural property, morphology, carbon source utilization, 16s rRNA gene analysis, and DNA-DNA relatedness studies. The anticancer component from L033 was purified to homogeneity by preparative positive-phase high-performance liquid chromatography and crystallization. Nuclear magnetic resonance and mass spectrometric analysis showed that this compound had the same structure as oligomycin A. Different with other reported naturally occurring strains of S. avermitilis, L033 produced high quantity of oligomycin A (maximal 1,461 μg/ml). Therefore, L033 was considered of great potential as an industrial oligomycin-A-producing strain.