The in vitro susceptibility to tobramycin of 1,466 bacterial isolates, including several genera and strains of common pathogenic gram-negative bacilli and Staphylococcus aureus, was determined by conventional serial dilution methods. Tobramycin exhibited marked in vitro antibacterial activity; almost all of the isolates tested were inhibited by less than or equal to 2.5 mug of the drug/ml. The antibiotic was used in the treatment of several types of infection in 29 patients with normal renal function. Observations were made on the therapeutic and secondary effects of the drug and on possible toxicity to the eighth cranial nerve. Clinical therapeutic results were satisfactory, The hearing of one patient was impaired, but the relation of this effect to tobramycin therapy is uncertain. Serum levels of the antibiotic were measured in 10 patients to investigate whether the drug accumulated during repeated administration. Mean serum levels of tobramycin 1 hr after intramuscular injection of 1.0 mg/kg were between 2.5 and 3.0 mug/ml on days 1, 3, and 6 of therapy. No accumulation of the antibiotic was observed after repeated administration.