Gentamicin acetyltransferase I is induced 13-fold in R factor resistant Escherichia coli by high concentrations (1 mg/ml) of gentamicin in the growth medium. The enzyme is maximally released from bacteria by osmotic shock in late-log phase, unlike previously studied periplasmic enzymes. Streptomycin sulfate and ammonium sulfate precipitations of shockate followed by affinity and ion-exchange chromatography recover 51% of the induced enzyme with a 360-fold increase in purity (12% of 4400-fold, uninduced). The purified enzyme appears homogeneous by six criteria, the first aminoglycoside inactivating enzyme so purified. Sodium dodecyl sulfate electrophoresis, amino acid analysis, and sedimentation analyses indicate a tetrameric protein of 63000 molecular weight. The protein does not contain tryptophan. Kinetic analyses yield apparent values of: Vmax = 3.4 +/- 0.2 mumol per min mg at pH 8 (optimum), Km (acetyl-CoA) = 3.9 +/- 0.2 muM, Km (gentamicin Cla) = 0.3 +/- 0.08 muM, KI (gentamicin substrate inhibition) = 160 +/- 29 muM. The activity of the enzyme is stable to a variety of conditions, including lyophilization and prolonged storage, and can be monitored by two convenient spectrophotometric assays.