An Escherichia coli strain with a plasmidic amikacin resistance has been selected for which the evidence strongly indicates that resistance is mediated by aminoglycoside phosphotransferase [APH(3')-II]: (i) this resistance was coupled with resistance against kanamycin and neomycin; (ii) partially purified APH(3')-II[APH(3") free] modified amikacin by phosphorylation; (iii) the product of the APH(3')-II mediated reaction (i.e., 3'-O-phosphoryl-amikacin) lost its antibacterial activity; and (iv) the amikacin-modifying APH(3')-II activity increased 5- to 10-fold after adaptation of the cells to higher concentrations of amikacin. The substrate spectrum of this enzyme showed a low activity against amikacin as compared with neomycin. It is argued that the enzyme level rather than its substrate spectrum is important for enzyme-mediated resistance. The increase in enzyme levels was found to be correlated with an increase in copy number of a 110-Megadalton plasmid (pBN66) which coded for the APH(3')-II and the APH(3") activity. The increase in copy number was irreversible, and therefore this phenomenon is ascribed to a mutation of a gene which affects the copy number. In transconjugants, the original low copy number was present, and therefore the mutation must be located on the chromosome and not on the plasmid.