The in vitro activity of the aminoglycoside antibiotic tobramycin was demonstrated by broth dilution and single-disc methods on 50 isolates each of Staphylococcus aureus, Klebsiella or Enterobacter, indole-positive and -negative Proteus, Escherichia coli, and Pseudomonas aeruginosa. All organisms were inhibited by 6.25 mug or less of the drug/ml. Pseudomonas strains resistant to kanamycin or gentamicin or both were susceptible to tobramycin. Those strains which were inhibited by 6.25 mug of tobramycin/ml by the broth dilution method had zone diameters of 16 mm or more by the single-disc method. Of 313 organisms tested by the disc method, 3 strains were found to be resistant to tobramycin, 73 were resistant to kanamycin, and 18 were resistant to gentamicin. Tobramycin was found to have satisfactory in vitro activity against many clinically important organisms, including strains resistant to gentamicin and kanamycin.