The murine immune response to Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease, is characterized by the development of antibodies reactive with the outer surface protein (Osp) A. It has been demonstrated that passive immunization of mice with at least some Osp A antibodies, including an Osp A mAb (CIII.78) that binds to a conformational epitope in the carboxyl-terminus of Osp A, provides protection against Bb challenge. Active immunization of mice with Osp A also confers protection, making Osp A a candidate for a vaccine Ag. To determine the regions of the Osp A protein that can elicit protective immunity, we immunized boosted mice with overlapping recombinant truncated fragments of Osp A, then challenged them with Bb. All groups of mice developed IgG Osp A antibodies detectable by immunoblotting with sera diluted at least 5000-fold. As expected, vaccination with full-length recombinant Osp A protected mice from challenge infection. In contrast, none of the mice vaccinated with the truncated Osp A proteins demonstrated immunity, even those immunized with an Osp A fragment binding the neutralizing mAb CIII.78. Osp A antibodies contained in the truncated Osp A antisera also failed to immunoprecipitate in vitro translated full-length Osp A and did not bind, as demonstrated by indirect immunofluorescence, to live or acetone-fixed Bb. Taken together, these results suggest that neutralizing Osp A antibodies are induced by vaccination with the full-length recombinant Osp A protein but not by vaccination with recombinant fragments.