The broad-spectrum aminoglycoside phosphotransferase, APH(3')-IIIa, confers resistance to several aminoglycoside antibiotics in opportunistic pathogens of the genera Staphylococcus and Enterococcus. The profile of the drug resistance phenotype suggested that the enzyme would transfer a phosphate group from ATP to the 3'-hydroxyl of aminoglycosides. In addition, resistance to the 3'-deoxyaminoglycoside antibiotic, lividomycin A, suggested possible transfer to the 5"-hydroxyl of the ribose [Trieu-Cuot, P., & Courvalin, P. (1983) Gene 23, 331-341]. Using purified overexpressed enzyme, we have prepared and purified the products of APH(3')-IIIa-dependent phosphorylation of several of aminoglycoside antibiotics. Mass spectral analysis revealed that 4,6-disubstituted aminocyclitol antibiotics such as amikacin and kanamycin are monophosphorylated, while 4,5-disubstituted aminoglycosides such as butirosin A, ribostamycin, and neomycin B are both mono- and diphosphorylated by APH(3')-IIIa. Using a series of one- and two-dimensional 1H, 13C, and 31P NMR experiments, we have unambiguously assigned the regiospecificity of phosphoryl transfer to several antibiotics. The 4,6-disubstituted aminocyclitol antibiotics are exclusively phosphorylated at the 3'-OH hydroxyl, and the 4,5-disubstituted aminocyclitol antibiotics can be phosphorylated at both the 3'- and 5"-hydroxyls. The first phosphorylation can occur on either the 3'- or 5"-hydroxyl group of neomycin B or butirosin A. Initial phosphotransfer to the 3'-position predominates for butirosin while the 5"-OH is favored for neomycin. These results open the potential for the rational design of aminoglycoside kinase inhibitors based on functionalization of either the 6-aminohexose or the pentose rings of aminoglycoside antibiotics.