Seven amikacin-resistant strains of Enterobacteriaceae isolated in Slovakia and Germany were included in this study. The strains were also resistant in vitro to high levels of gentamicin, tobramycin, netilmicin and isepamicin. Phosphocellulose paper binding assays indicated that resistance to aminoglycosides was due to synthesis of aminoglycoside acetyltransferase AAC(6′)-I in combination with aminoglycoside phosphotransferase APH(2XXX), a mechanism until now only identified in staphylococci and streptococci. This mechanism of aminoglycoside resistance has also been found in two isolates of Klebsiella pneumoniae from Germany. The substrate profile suggested that in addition to AAC(6′)-I and APH(2XXX), several strains also produced AAC(3)-II. Aminoglycoside resistance was found to be transferable to Escherichia coli 3110 rifr in all isolates, and R plasmids of 36-45 MD were detected in donor and transconjugant strains. All isolated plasmids from transconjugants encoded resistance to aminoglycosides by genes encoding the enzymes AAC(6′)-I and APH(