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Objectives Multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa have become a global problem, often leaving the polymyxins as therapeutic agents of last resort. ACHN-490, a next-generation aminoglycoside with activity against a broad range of Gram-positive and Gram-negative pathogens, was examined against clinical isolates of A. baumannii and P. aeruginosa. Methods The activity of aminoglycosides and ACHN-490 was determined against a contemporary collection of A. baumannii and P. aeruginosa. Selected aminoglycoside-resistant isolates were screened for the presence of genes encoding common aminoglycoside-modifying enzymes and methylases. Results Resistance to the traditional aminoglycosides was common in the collection of A. baumannii. ACHN-490 possessed superior activity against these isolates, with an MIC50 value of 8 mg/L. In P. aeruginosa, the activity of ACHN-490 was similar to that of amikacin (MIC50 value of 8 mg/L for both agents). For both A. baumannii and P. aeruginosa, the MICs of ACHN-490 did not correlate with the presence of commonly encountered aminoglycoside-modifying enzymes. Conclusions For A. baumannii, the MICs of ACHN-490 were lower than those of traditional aminoglycosides. For P. aeruginosa, the activity of ACHN-490 was similar to that of amikacin. [ABSTRACT FROM PUBLISHER]