Gentamicin resistance gene residing in Serratia marcescens R plasmid pIS199G2 was cloned after it was transferred to Escherichia coli ML1410 by conjugation. Partial digests of pIS199G2 with Sau3A1 were cloned into the BamHI site of pUC9. One plasmid pSY1 carried 1.5kb insert and showed gentamicin resistant phenotype. A polypeptide product of 31kd was identified from pSY1 in minicell expression system. During the cloning procedure a plasmid naboring a replication origin in R plasmid, ampicillin resistance gene and gentamicin resistance gene was obtained. The plasmid, pSW1, also produced the polypeptide of the same size by the minicell expression system. The restriction enzyme mapping of pSY1 and pSW1 showed homology to other aacC2 type of gentamicin resistance gene and suggests that the two plasmids contain aacC2 gene family.