An in vitro assay was used to characterize the borreliacidal activity of sera from Lyme disease patients. The mean percentage of killing was 23% with sera from patients with a single erythema migrans lesion, 42% from patients with multiple lesions, 58% from patients with Lyme arthritis of short duration, and 83% from patients with Lyme arthritis of long duration. Borreliacidal activity was abrogated when Lyme disease serum was treated with anti-human IgM or IgG1. In addition, human sera from Lyme arthritis patients containing borreliacidal antibody prevented the induction of Lyme arthritis in irradiated hamsters challenged with the Lyme spirochete. Removal of outer surface protein A antibodies from late Lyme disease sera caused reductions in the borreliacidal antibody titer. The results demonstrate an important role for borreliacidal antibody against infection with B. burgdorferi in humans and confirm that detection of borreliacidal antibody in human sera can be a specific serodiagnostic test for Lyme disease.