In this work, hydrogen-deuterium exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3 '-IIIa (APH), a protein of intense interest due to its involvement in conferring antibiotic resistance to both Gram negative and Gram positive microorganisms. This represents the first characterization of dynamic properties of an aminoglycoside-modifying enzyme. Herein we describe in vitro dynamics of apo, binary, and ternary complexes of APH with kanamycin A, neomycin B, and metal-nucleotide. Regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 h of exposure to D2O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange, and similar to 30% of the amides remain unexchanged up to 95 h after exposure to D2O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to the enzyme-aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby beta-sheet, thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide a molecular basis for broad substrate selectivity of APH.