NMR spectroscopy has been used to study the interaction of aminoglycoside antibiotics with an aminoglycoside antibiotic 3'-phosphotransferase [APH(3')-IIIa]. APH(3')-IIIa is an enterococcal enzyme that is responsible for the ATP-dependent O-phosphorylation of a broad range of aminoglycoside antibiotics. The NMR method of transferred nuclear Overhauser effect spectroscopy (TRNOESY) was used to detect intra- and inter-ring NOEs for butirosin A and amikacin in their respective ternary complexes with APH(3')-IIIa and ATP. NOE-derived distance constraints were used in energy minimization and dynamics routines to yield enzyme-bound structures for butirosin A. These structures suggest that the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings have restricted motions and are in a stacking arrangement. The TRNOE spectra for amikacin suggest that the 6-amino-6-deoxy-D-glucose ring is flexible when the antibiotic is bound to APH(3')-IIIa. The 15N resonances of butirosin A were assigned and the pKa values of the amino groups of butirosin A and amikacin were determined by 15N NMR spectroscopy. The N3 amino groups of butirosin A and amikacin have lowered pKa values, which is attributed to the (S)-4-amino-2-hydroxybutyryl (AHB) group of the antibiotics. This work provides an insight into the geometrical and electrostatic nature of aminoglycoside antibiotics bound to a modifying enzyme and will provide a basis for the design of inhibitors of APH(3')-IIIa.