The 41-kD antigen of Borrelia burgdorferi is an immunodominant protein that is recognized early by antibodies in sera from Lyme disease patients and known to be associated with the endoflagella. We identified the 41-kD endoflagellar antigen to be a single polypeptide with an apparent isoelectric point (pI) of 6.5 by two-dimensional (2-D) electrophoresis. This polypeptide, which we designated P41F alpha, was heavily labeled by 125I in 2-D autoradiographs of B. burgdorferi whole-cell lysates and was recognized by a murine monoclonal antibody (MCB1) and human antisera in 2-D immunoblots. NH2-terminal sequence analysis showed 80% homology between P41F alpha and the 33-kD endoflagellar protein of Treponema pallidum. Results of indirect immunofluorescence assays (IFA), Triton X-114 phase partitioning, and agglutination studies suggested a possible surface exposure of the polypeptide. Silver stained 2-D gels also revealed the presence of another 41-kD species, with an apparent pI of 6.6 (designated P41 beta), which was not radioiodinated in 2-D autoradiographs, and was not recognized by MCB1 or human antisera. NH2-terminal sequence analysis of P41 beta revealed no homology with P41F alpha, leading to the conclusion that they are not related.