Current biological and serological techniques for demonstrating infections by Borrelia burgdorferi can be inconclusive. In order to monitor Lyme borreliosis, we developed a rapid and sensitive assay for B. burgdorferi antigens in infected hosts. Polyclonal rabbit antisera were raised against membrane vesicles and an 83-kDa vesicle-associated protein band that was purified from in vitro B. burgdorferi cultures. Immunoglobulin G (IgG) antibodies were recovered from these sera and tested for a species-specific reaction with several geographically diverse Borrelia isolates by immunoblot analysis. Parlodion-coated electron microscope grids were activated with anti-vesicle F(ab')2 fragments and then incubated with confirmed or experimental sources of spirochetal antigens. Such sources included cultured spirochetes; spirochete culture supernatants; samples of urine, blood, or serum from mice, dogs, and humans; triturates of Ixodes ticks; and bladder, spleen, liver, kidney, heart, or brain tissues from infected or control mice. Captured antigens were assayed by immune electron microscopy by using anti-83-kDa IgG antibodies and protein A-colloidal gold conjugates. The results indicated that B. burgdorferi appears to shed surface antigens which are readily detectable in urine, blood, and several organs from infected hosts. Such antigens were detectable in mouse urine at dilutions exceeding 10(-6). Intact spirochetes were frequently observed on grids incubated with blood, spleen, or bladder preparations, and B. burgdorferi was reisolated from the urinary bladders of all experimentally infected mice. These results indicated that B. burgdorferi antigens arise in a variety of host materials. Such antigens can be captured and identified with specific polyclonal antibodies, providing a sensitive assay for monitoring and studying Lyme borreliosis.