Gene for aminoglycoside 6'-N-acetyltransferase [AAC(6')] from Serratia sp. 45 was cloned into E. coli. The enzyme produced in E. coli carrying the recombinant plasmid was compared to the Serratia enzyme. Both enzymes acetylated the 6'-C position of amikacin, dibekacin, tobramycin, sisomicin, gentamicin C1a and kanamycin but effected gentamicin C1, gentamicin C2 and micronomycin minimally. No significant difference in optimal pH, isoelectric point or molecular weight was detected. The nucleotide sequence of the gene was determined. Initiating with a GTG codon for methionine, it was composed of 552 base pair coding for 184 amino acids. The molecular weight of the enzyme was about 20418. Comparison of the amino acid sequence of this AAC(6') with the amino acid sequence of aacA4 gene from Serratia marcescens (G. Tran Van Nhieu and E. Collatz, J. Bacteriol., 169, 5708(1987)) showed 98.3% homology.