An effective public health response to a deliberate release of Bacillus anthracis will require a rapid distribution of antimicrobial agents for postexposure prophylaxis and treatment. However, conventional antimicrobial susceptibility testing for B. anthracis requires a 16- to 20-h incubation period. To reduce this time, we have combined a modified broth microdilution (BMD) susceptibility testing method with real-time quantitative PCR (qPCR). The growth or inhibition of growth of B. anthracis cells incubated in 2-fold dilutions of ciprofloxacin (CIP) (0.015 to 16 microg/ml) or doxycycline (DOX) (0.06 to 64 microg/ml) was determined by comparing the fluorescence threshold cycle (C(T)) generated by target amplification from cells incubated with each drug concentration with the C(T) of the no-drug (positive growth) control. This DeltaC(T) readily differentiated susceptible and nonsusceptible strains. Among susceptible strains, the median DeltaC(T) values were > or = 7.51 cycles for CIP and > or = 7.08 cycles for DOX when drug concentrations were at or above the CLSI breakpoint for susceptibility. For CIP- and DOX-nonsusceptible strains, the DeltaC(T) was < 1.0 cycle at the breakpoint for susceptibility. When evaluated with 14 genetically and geographically diverse strains of B. anthracis, the rapid method provided the same susceptibility results as conventional methods but required less than 6 h, significantly decreasing the time required for the selection and distribution of appropriate medical countermeasures.