Netilmicin (Sch 20569), a semisynthetic aminoglycoside antibiotic, was compared with gentamicin, tobramycin, and amikacin against 242 clinical isolates of Pseudomonas and Enterobacteriaceae. The minimum inhibitory concentration (MIC) was determined in both solid and liquid media. Netilmicin exhibited typical aminoglycoside properties, such as little effect of inoculum size on MIC, relatively small gap between MIC and minimum bactericidal concentration, and potentiation of anti-Pseudomonas activity in the presence of carbenicillin. Netilmicin provided no advantage in antimicrobial activity over gentamicin for either Pseudomonas or Enterobacteriaceae. Nearly complete cross-resistance to netilmicin was encountered with isolates resistant to gentamicin in either solid or liquid media. Netilmicin was less active than gentamicin against isolates of Pseudomonas and Providencia. Major discrepancies between MIC values determined in agar as opposed to those determined in broth were encountered for most isolates of Pseudomonas but also, depending upon antibiotic tested, for between 15 and 40% of isolates of Enterobacteriaceae. This new aminoglycoside agent will be useful clinically only if it is shown to be significantly less toxic than presently available analogues.