The arrangements of the antibiotics amikacin and butirosin A at the active site of an aminoglycoside antibiotic 3’-phosphotransferase (APH(3’)-IIIa), which mediates resistance to a broad spectrum of aminoglycoside antibiotics, were determined. APH(3’)-IIIa phosphorylates a wide range of aminoglycoside antibiotics in an ATP-dependent manner. beta,gamma-Bidentate CrATP, a stable exchange-inert metal-nucleotide analog, was used as a paramagnetic probe to determine the arrangement of amikacin and butirosin A in the respective enzyme CrATP antibiotic complexes. The paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T-1p) of the H-1 nuclei of amikacin and butirosin A were examined to determine the distances between enzyme-bound CrATP and various protons of these aminoglycoside antibiotics in the ternary APH(3’)-IIIa CrATP antibiotic complexes. From these distances, models were constructed that represent possible enzyme-bound arrangements and conformations for these aminoglycosides. These models show that amikacin and butirosin A adopt different arrangements at the active site of APH(3’)-IIIa. The results for butirosin A suggest that the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings are in a stacking arrangement which is consistent with its solution structure. This is the first paper to describe the arrangement and conformation of aminoglycoside antibiotics bound to a modifying enzyme and is an important step in the design of novel antibiotics and/or enzyme inhibitors.