An aminoglycoside-acetylating enzyme produced by a strain of Escherichia coli with an unusual resistance phenotype was characterized. This enzyme was found to mono-acetylate apramycin, butirosin, lividomycin and paromomycin and diacetylate ribostamycin and neomycin to give reaction products which were distinguishable by HPLC analysis from those of AAC(2'), AAC(3) and AAC(6') enzymes. The enzyme, however, was not found to acetylate amikacin, fortimicin, geneticin, gentamicin, kanamycin A, netilmicin or tobramycin. The reaction product from the action of this enzyme on apramycin was purified and identified by nuclear magnetic resonance studies as 1-N-acetyl apramycin. The second site at which ribostamycin and neomycin B were modified by this enzyme was not determined but is postulated as the 6'-amino group. It is proposed that this enzyme be named AAC(1).