Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log2 dilution higher in CO2 than in AA; tetracycline-class MICs to be 1 log2 dilution lower in CO2.