Real-time polymerase chain reaction (PCR) assays were developed to detect Francisella tularensis (Ft), the causative agent of tularaemia in humans. Two real-time PCRs (FTT0376 and FTT0523) were designed in genetic sequences identified by the Insignia genome comparison tool (http://insignia.cbcb.umd.edu/) as being unique to pathogenic subspecies of F. tularensis. Both PCRs identified all pathogenic F. tularensis subspecies but did not cross react with avirulent Francisella philomiragia or F. tularensis ssp. novicida or other environmental bacteria. Limits of detection from DNA purified from pure culture (FTT0376 approximately 80 Ft genome equivalents (GEs) per PCR; FTT0523 approximately 20 Ft GEs per PCR;) and DNA purified from spiked blood samples (4 x 10(4) to 4 x 10(3) cfu ml(-1), both assays) were determined.