The in vitro activity of tobramycin was studied against 457 clinical isolates of gram-negative bacilli and 151 clinical isolates of gram-positive cocci. The vast majority of the gram-negative bacilli was inhibited by tobramycin at a concentration of 1.56 μg or less per ml. Only a few isolates of Staphylococcus aureus and no isolates of Streptococcus pyogenes or Diplococcus pneumoniae were susceptible to this drug. Tobramycin was generally more active than gentamicin sulfate against gram-negative bacilli, although organisms resistant to gentamicin sulfate were also resistant to tobramycin. The major difference between the two drugs was the greater activity of tobramycin against Pseudomonas aeruginosa .