The soluble and ionized calcium and magnesium contents of 18 lots of Mueller-Hinton agar medium from three different manufacturers were analyzed, and the results were correlated with medium performance. A standardized disk diffusion test, with Pseudomonas aeruginosa (ATCC 27853) and a 10-microgram gentamicin disk, served as an indicator of medium performance. Zone diameters correlated well with the ionized calcium values and the sum of the ionized calcium and soluble magnesium values in the different lots (r = -0.88 for both). Zone diameters correlated poorly with ionized magnesium values (r = -0.57), which were best described by a curvilinear relationship. Supplementation of lots of Mueller-Hinton agar medium with equivalent amounts of calcium and magnesium as the chloride, gluconate, or glycerophosphate salts produced identical decreases in zone sizes. Adjustment of deficient lots of Mueller-Hinton agar medium with ionized calcium or soluble magnesium or both (as the gluconate salts), to match the concentrations in lots that provided satisfactory zone sizes (17 to 19 mm), resulted in performance comparable to that of the control lots. Sixteen strains of Pseudomonas aeruginosa, ranging from resistant to susceptible, responded to cation adjustment in the same manner as the ATCC quality control strain. Satisfactory medium performance can obviously be assured by biological means in aminoglycoside susceptibility testing of Pseudomonas aeruginosa on Mueller-Hinton medium; however, cation adjustment of medium to predetermined levels of ionized calcium and soluble magnesium can now also provide desirable performance levels for P. aeruginosa on Mueller-Hinton medium.