Standard minimum inhibitory and bactericidal concentrations are not established for most antimicrobial agents against strains of bacteria commonly used for quality control in susceptibility testing. The effects of cation and human serum supplementation of broth on the values are also unknown. Therefore, we performed 10 minimum inhibitory and bactericidal concentration determinations for 44 antimicrobial agents against the standard control strains Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853 in Mueller-Hinton broth and in Mueller-Hinton broth supplemented with calcium, magnesium, and 50% pooled human serum. Agreement of replicates was within one twofold dilution 97% of the time. Supplemented Mueller-Hinton broth gave higher minimum inhibitory concentrations for 24 antibiotics against S. aureus, for 17 drugs against E. coli, and for 12 drugs against P. aeruginosa, whereas it gave lower minimum inhibitory concentrations for 1 antibiotic against S. aureus, for 5 against E. coli, and for 5 against P. aeruginosa. Results for minimum bactericidal concentrations were similar. Added serum did not further affect the increased resistance of P. Aeruginosa to aminoglycosides encountered with cation supplementation of broth. These results provide expected values for the quality control strains when minimum inhibitory and bactericidal concentrations are determined in these two Mueller-Hinton media.