The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3')-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal alpha-helix that contains the COOH-terminal residue Phe(264). We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3')-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp(261), Glu(262), and Phe(264)), and two additional residues that line the wall of the anionic depression (Tyr(55) and Arg(211)). Using a molecular modeling approach, six ternary complexes of APH(3')-IIIa.ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe(264) is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.