A genomic DNA library of Pseudomonas paucimobilis TMY1009 was constructed using a cosmid vector pWE15. Screening of the library for lignostilbene-alpha,beta-dioxygenase (LSD) isozyme genes was done with a common probe for the alpha, beta, and gamma subunits, which composed LSD isozymes. The positive clones obtained by colony hybridization were further confirmed by Southern hybridization. A 4.2-kb BamHI-HindIII fragment hybridized with the probe was subcloned into pUC118 to yield the plasmid pKHE1700. Escherichia coli MV1184 carrying pKHE1700 produced one of the LSD isozymes. The cloned LSD was purified and compared with LSD isozymes from P. paucimobilis TMY1009. The behavior on column chromatographies through all purification steps accorded with that of LSD-III. Furthermore, the mobility on polyacrylamide gel electrophoresis, the elution profile on reversed-phase HPLC, and the partial amino acid sequence were common to the cloned LSD and the native LSD-III. Thus the cloned LSD could be identified as LSD-III. It was found that the gene of LSD-III (lsdB) was composed of 489 codons.