Borreliacidal antibody has been shown to be important for the serodiagnosis of Lyme disease and determination of immune status. Our results show that borreliacidal antibody can be rapidly and accurately detected by flow cytometry. Acridine orange was added to normal and immune sera containing Borrelia burgdorferi organisms in the presence and absence of complement prior to data acquisition by flow cytometry. The flow cytometric parameters of side scatter and detection of acridine orange fluorescence were used to determine events per minute (number of labeled spirochetes), percent shift in fluorescence (number of dead spirochetes), and mean channel fluorescence (intensity of fluorescence-labeled spirochetes) of acridine orange-labeled spirochetes. Borreliacidal antibody was detected as early as 4 h, with optimal detection 16 to 24 h after incubation of B. burgdorferi organisms with immune serum and complement. Our results also showed that complement was necessary for detection of borreliacidal antibody. Flow cytometry with acridine orange-labeled spirochetes provides a rapid means for detection of borreliacidal antibody.