Many antibiotics contain partially deoxygenated sugar components that are usually essential for biological activity, affinity, structural stability, and solubility of antibiotics. Gene probes of the biosynthetic genes related with the deoxysugar were obtained from PCR. Primers were designed from the conserved peptide sequences of the known dTDP-D-glucose 4,6-dehydratases, which are the key step enzymes in the biosynthesis of deoxysugar. The primers were applied to amplify parts of dehydratase genes to 27 actinomycetes that produce the metabolites containing deoxysugar as structural constituents. About 180 and 340 bp DNA fragments from all of the actinomycetes were produced by PCR and analyzed by Southern blot and DNA sequencing. The PCR products were used as gene probes to clone the biosynthetic gene clusters for the antibiotic mithramycin, rubradirin, spectinomycin, and elaiophyrin. This method should allow for detecting of the biosynthetic gene clusters of a vast array of secondary metabolites isolated from actinomycetes because of the widespread existence of deoxysugar constituents in secondary metabolites.