Gentamicin accumulation with time shows multiphasic kinetics in strains of Escherichia coli and Pseudomonas aeruginosa. All but first phase accumulation may be prevented or reduced by inhibitors of electron transport, by a sulfhydryl poison, by agents which uncouple electron transport and oxidative phosphorylation and by an inhibitor of protein synthesis. The phases of accumulation which are sensitive to these inhibitors are required for loss of cell viability. Gentamicin can be extracted from cells in an unchanged form as judged by paper chromatography and is concentrated 4 to 250 times over extracellular concentrations within the bacterial cell. Gentamicin accumulation has been shown to occur before there is any evidence or release of acid-soluble 3H-adenine from cells. These data demonstrate that productive gentamicin accumulation capable of causing cell death is by active transport.