Fifty-five strains of Pseudomonas aeruginosa were tested against arithmetic increments in concentrations of gentamicin, tobramycin, and amikacin in 14 different lots of Mueller-Hinton agar. The divalent cation content of each lot was determined by atomic absorption spectrometry. The relation between mean minimal inhibitory concentration (MIC) for strains within each lot and cation content was studied by stepwise regression. Among the cations, the content of Zn++ most highly correlated with the MIC of each aminoglycoside; however, Zn++ accounted for only 23%, 60%, and 47% of the variability in the mean MIC of gentamicin, tobramycin, and amikacin, respectively, against all strains. In two-cation models Zn++ with Ca++ or Cu++ was most highly correlated with the mean MICs of the three aminoglycosides against all strains. No divalent cation, either singly or in combination with one or two other cations, gave a good prediction of the MICs of the aminoglycosides in agar. Furthermore, there was variability in the cations that most highly correlated with the MICs for some strains. These observations support the concept that ionic strength, cations, and a variety of other as yet poorly defined components of media influence the activity of aminoglycosides against P. aeruginosa.