The resistance to aminoglycoside antibiotics in Staphylococcus epidermidis FK109, is mediated by an enzyme that catalyzes transfer of the nucleotide monophosphate moiety from the nucleotide triphosphates, either to the 4'-hydroxyl group, that is in the equatorial plane of the aminoglycoside molecule. The enzyme, modifying the two sites, appears as a single and homogeneous entity in affinity chromatography, in chromatography on DEAE-Sepharose CL-6B, in isoelectric focusing and in gel-filtration. It requires divalent cations, notably Mg++, and dithiothreitol for optimal adenylylation. It has a molecular weight of 46,770 and an isoelectric point of 5.0. The ability of the enzyme ANT (4', 4'') to modify the two hydroxyl groups of aminoglycoside molecules, enables it to have a spectrum of substrates that surpasses, in range, the substrate spectrum of all the aminoglycoside-modifying enzymes which have been characterized hitherto.