Background: Borreliacidal antibodies can be detected in serum samples from patients with early or late Lyme disease symptoms. When these serum samples are incubated with Borrelia burgdorferi and complement, spirochetes are rapidly killed. Detection of these antibodies can be used as a serodiagnostic test. Methods: Individual serum samples containing IgM or IgG borreliacidal antibodies were used to develop a method for detection using flow cytometry. An additional 10 case-defined Lyme disease serum samples and 10 normal serum samples were used to confirm appropriate flow cytometric parameters. To determine specificity, 157 normal serum samples and 104 potential cross- reactive serum samples were tested for borreliacidal activity and antibodies to B burgdorferi using indirect fluorescent antibody or enzyme immunoassay. Results: Flow cytometry can be used to detect borreliacidal activity within 16 to 24 hours after incubation of B burgdorferi organisms, Lyme disease serum, and complement. Significant borreliacidal activity was detected in all Lyme disease serum samples. The percentages of positive normal serum samples were comparable (6% to 10%) using all three assays. In addition, the indirect fluorescent antibody and enzyme immunoassay identified 41 (39%) and 47 (45%) potential cross-reactive serum samples as positive, respectively. In contrast, significant borreliacidal activity was not detected in any potential cross-reactive serum samples. Conclusion: Detection of borreliacidal antibody, unlike indirect fluorescent antibody and enzyme immunoassay, is an accurate, highly specific serodiagnostic test for detection of Lyme disease.