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A 50-kb chromosome DNA region was isolatedfrom Streptomyces kanamyceticus by screening the fosmidgenomic library, using the 16S rRNA methylase gene (kmr)as a probe. Sequence analysis of this region revealed 42putative open reading frames (ORFs), which included biosyntheticgenes such as genes responsible for 2-deoxystreptamine (2-DOS) biosynthesis as well as genes for resistance and regulatoryfunction. Also, the kanamycin acetyltransferase gene (kac)was characterized by in vitro enzyme assay, which conferredE. coli BL21 (DE3) with 10, 50, and 80-times higher resistanceto kanamycin A, tobramycin, and amikacin, respectively, thanthe control strain had, thus strongly indicating that the isolatedgene cluster is very likely involved in kanamycin biosynthesis.This work provides a solid basis for further elucidation of thekanamycin biosynthesis pathway as well as the productivityimprovement and construction of new hybrid antibiotics.