A modified Mueller-Hinton broth was developed to perform antimicrobial susceptibility tests on Francisella tularensis. Adequate growth of the organism was obtained within 24 h of inoculation, and MICs could be read at that time. We tested 15 selected strains of F. tularensis and five reference quality control strains in this medium with 36 antimicrobial agents. The MICs of the aminoglycosides and tetracycline increased 1 to 3 dilutions in this medium compared with those in the usual medium, but the other antimicrobial agents were not consistently affected by the medium. Even though the medium caused an increase in MICs, the aminoglycosides and tetracyclines remained very active in vitro against F. tularensis. Other antimicrobial agents effective in vitro were chloramphenicol, erythromycin, ceftazidime, moxalactam, cefotaxime, ceftriaxone, and Sch 29482 (a cephalosporin).