The antimicrobial activity of netilmicin, a new semisynthetic aminoglycosidic aminocyclitol, was determined against 123 recent gram-negative clinical isolates susceptible to gentamicin and 60 isolates resistant to either sisomicin, gentamicin, or tobramycin. The minimal inhibitory concentrations and minimal bactericidal concentrations of netilmicin, sisomicin, gentamicin, and tobramycin against Pseudomonas, Escherichia coli, Klebsiella, Enterobacter, Proteus mirabilis, and indole-positive Proteus were, in general, quite similar. Gentamicin was the most active against Serratia. A total of 54, 67, and 88% of gentamicin-resistant Pseudomonas, Serratia, and Klebsiella, respectively, were susceptible to netilmicin. Strains of indole-positive Proteus, Acinetobacter, Providencia, and E. coli resistant to gentamicin were likely to be resistant also to netilmicin.