The in vitro activity of netilmicin was compared to gentamicin, tobramycin and amikacin against 461 strains of glucose fermenting and nonfermenting bacteria. The minimum inhibitory concentrations of netilmicin, gentamicin and tobramycin against the majority of Enterobacteriaceae, Staphylococcus and Streptococcus were quite similar. Gentamicin, however, was approximately fourfold more active against strains of S. marcescens. Amikacin was the most effective antibiotic against strains of fermenting and nonfermenting bacilli resistant to at least one aminoglycoside. Many gentamicin-resistant species of nonfermenting bacilli, however, remain highly resistant to all four aminoglycosides tested.