Background: Tularemia is an infection caused by Francisella tularensis, which has a wide distribution in the northern hemisphere and diverse clinical manifestations. For decades, the drug of choice for the treatment of tularemia has been streptomycin, with tetracycline and chloramphenicol being used as alternatives. The purpose of the present study was to determine the in vitro antimicrobial susceptibility of a large panel of geographically diverse F. tularensis isolates from Turkey against traditional and newer antimicrobial agents. Methods: The antibiotic susceptibilities of 250 F. tularensis strains were examined using the Epsilometer test for 9 antimicrobial agents. Each isolate was identified by conventional and molecular techniques. Results: All the strains were confirmed biochemically and using a combination of species- and subspecies-specific polymerase chain reaction (PCR) assays to be F. tularensis subsp. holarctica. One isolate was assigned to F. tularensis subsp. holarctica biovar japonica based on erythromycin susceptibility, an ability to ferment glycerol, and the nucleotide sequence of the region of difference 1 (RD1). All strains were susceptible to aminoglycosides (streptomycin and gentamicin), tetracyclines (tetracycline and doxycycline), chloramphenicol, 2 fluoroquinolones (ciprofloxacin and levofloxacin), and rifampicin. In addition, all isolates except 1 had a minimal inhibitory concentration (MIC) for erythromycin of >256 mu g/ml. Conclusions: Since the fluoroquinolones showed the lowest MIC values and have advantages such as excellent bioavailability and activity, availability of oral formulations, and lower toxicities, they represent candidate therapeutic options in the first-line treatment of tularemia. To the best of our knowledge, this is the first report of the presence of F. tularensis subsp. holarctica biovar japonica outside Japan.