The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.