Genes encoding aminoglycoside 6′- N -acetyltransferases, were identified using the polymerase chain reaction (PCR). Four sets of primers delineating DNA fragments of 209 bp, 250 bp, 260 bp and 347 bp, specific for the four known aacA genes, and probes within these fragments, were constructed based on the nucleotide sequences of the aacA genes. The specificity of the primers was evaluated using reference strains encoding various aminoglycoside-modifying enzymes. The primers reacted with their corresponding aacA genes and did not cross-react with genes coding for other aminoglycoside-modifying enzymes. One hundred and sixty-one aminoglycoside resistant clinical isolates showing AAC(6′)I activity were tested using the PCR assays. The gene described by Tran Van Nhieu & Collatz (1987) was the most frequently identified aacA gene. One strain of Citrobacter freundii contained two distinct aacA genes. However, in 46% of the strains, the majority being Serratia spp. and Acinetobacter spp. none of the specific amplified DNA fragments for any of the known aacA genes could be detected.