Kanamycin resistance gene (kmr) from the overproducing mutant strain of Streptomyces kanamyceticus ISP1375 (strain 1) and from its gentamicin-resistant mutant were cloned into the high copy number vector pIJ702 and transformed into S. lividans 66. This gene provides resistance to kanamycin, gentamicin, sisomicin and tobramicin. The resistance of the transformed recipient strains was higher than the resistance level of the donor S. kanamyceticus 1. Sequencing of the kmr gene (EMBL Nucleotide Sequence Database accession no. Y15838 revealed 53.9% identity in 274 aa with the kgmB gene product (16S rRNA methylase) of S. tenebrarius. Hybridisation analysis using a 0.85 kb fragment carrying the kmr gene revealed that other gentamicin-resistant mutants of S. kanamyceticus 1 and unstable kanamycin-nonproducing mutant had a high level of kmr amplification. We found no homology between the kmr gene and the total DNA of the neomycin producer S. fradiae IFO3718; the sisomicin producer M. zionensis IFO14116 and the gentamicin producer M. purpurea ATCC15835.