Enzymatic phosphorylation of amikacin has not been reported previously in gram-negative bacteria. We found that extracts of MP1, a mutant of Escherichia coli JR66/W677 that is resistant to amikacin, were able to phosphorylate this aminoglycoside more rapidly than were extracts of the parental strain. Conjugal transfer of resistance from MP1 to a recipient strain was accompanied by acquisition in the transconjugants of amikacin phosphotransferase activity and of a 57-megadalton plasmid present in the donor. Partial purification of the phosphotransferase activity on amikacin-Sepharose 4B yielded an enzyme with a substrate spectrum similar to that of the 3'-neomycin-kanamycin phosphotransferase II found E. coli, except that it was also active against amikacin. A mutant of MP1, MP5, had increased susceptibility to amikacin and reduced phosphotransferase activity. MP9, a mutant MP5, was more resistant to amikacin and had increased phosphotransferase activity. The mutations leading to these alterations of amikacin susceptibility and amikacin phosphotransferase activity were transferable with the same plasmid that was associated with amikacin resistance and phosphotransferase activity in MP1. These studies demonstrate that resistance to amikacin in a laboratory strain of E. coli is due to an aminoglycoside phosphotransferase coded by a transferable plasmid-borne gene.