Patients with early Lyme disease produce IgM antibodies to a 41,000-dalton polypeptide of Borrelia burgdorferi. With advancing disease, IgG antibodies recognize a larger number of spirochetal polypeptides. Sodium dodecyl sulfate treatment of spirochetes at a concentration of 0.03% resulted in an outer envelope (OE) fraction in the supernatant and a protoplasmic cylinder (PC) flagellar fraction in the pellet. Shearing of the PC pellet followed by ultrafiltration and dialysis resulted in a flagellin-enriched fraction. From the OE and flagellin-rich fractions, individual polypeptides with Mr of 66,000, 41,000, and 31,000/34,000 daltons were eluted in microgram quantities after electrophoresis. The flagellin-enriched fraction as well as the eluted polypeptides were used as antigens in the ELISA. The flagellin-enriched fraction was superior to the standard ELISA antigen. The 41-kilodalton (kDa) flagellin antigen was not useful. Antigens of 66 kDa and 31/34 kDa were confirmed to be part of the late antibody response.