The antibacterial activity of four aminoglycoside antibiotics (gentamicin, Sch 13706, tobramycin, and sisomicin) was tested against eight gram-negative and three gram-positive species. A total of 323 strains were studied by the broth dilution technique. Tobramycin and sisomicin had greater bacteriostatic and bactericidal activity against Pseudomonas strains than did gentamicin and Sch 13706. Of the four antibiotics, sisomicin was most active against Klebsiella, Enterobacter, Escherichia coli, indole-negative and -positive Proteus, and Streptococcus pyogenes. Gentamicin was most effective against Serratia. A fourfold or greater difference existed frequently between the minimal inhibitory and bactericidal concentrations of all antibiotics against Enterobacter and Serratia. This difference was greatest with tobramycin. Staphylococcus aureus was highly susceptible, Providencia relatively resistant, and enterococcus uniformly resistant to the antibiotics studied. Agar diffusion susceptibility testing with gentamicin and tobramycin showed that organisms susceptible to less than 6.2 mug/ml usually yielded zones 17 to 26 mm in diameters. Zones of 15 to 16 mm represented intermediate susceptibility which varied with the organism and antibiotic. Several Serratia strains required 6.2 to 12.5 mug of gentamicin/ml or 25 to 50 mug of tobramycin/ml for bactericidal activity despite minimal inhibitory concentrations of 0.09 to 3.1 mug/ml and zone sizes greater than 13 and 17 mm, respectively. Studies with Enterobacter and tobramycin yielded similar results.