The in vitro activity of netilmicin (Sch 20569), a new semisynthetic derivative of gentamicin, was compared with that of gentamicin and amikacin. One hundred and ninety-two clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus were tested using both agar and broth dilution techniques. Netilmicin was comparable to gentamicin, with the following exceptions: (i) for Serratia marcescens and P. aeruginosa, gentamicin was more active than netilmicin; (ii) all strains of Escherichia coli, Klebsiella, Enterobacter, Proteus mirabilis, and Citrobacter freundii, which were resistant to gentamicin, were susceptible to netilmicin; (iii) some strains of S. marcescens, indole-positive Proteus, and Providencia, which were resistant to gentamicin, were susceptible to netilmicin. Netilmicin was more active than amikacin for all Enterobacteriaceae and S. aureus and equal to amikacin in activity against gentamicin-susceptible strains of P. aeruginosa. All strains of P. aeruginosa, resistant to gentamicin, were also resistant to netilmicin but were susceptible to amikacin. Minimal inhibitory concentrations (MICs) obtained with broth and agar showed no significant differences except for P. mirabilis, where broth MICs were twofold greater than agar MICs, and for P. aeruginosa, where agar MICs were twofold higher than broth MICs. The minimal bactericidal concentration (MBC) was either identical to or within one twofold dilution of the MIC for the strains tested. A 100-fold increase in inoculum size produced less increase in MIC and MBC with netilmicin than with gentamicin or amikacin.