BmpA (P39) is an immunodominant chromosomally encoded Borrelia burgdorferi protein. The potential strong cross-reactivity of anti-BmpA antibodies with the other members of this paralogous protein family and the previous use of antibodies whose reactivity to the other Bmp proteins was uncharacterized have resulted in continued controversy over its localization in B. burgdorferi. In an effort to provide a definitive demonstration of the localization of BmpA, rabbit antibodies raised to recombinant BmpA (rBmpA) were rendered monospecific by absorption with rBmpB. This reagent did not react with rBmpB, rBmpC, or rBmpD in dot immunobinding, detected only a single 39-kDa band and a single 39-kDa, pI 5.0 spot on one- and two-dimensional immunoblots of B. burgdorferi lysates, respectively, and immunoprecipitated a single 39-kDa protein from these lysates. It detected BmpA in the Triton X-114-soluble and -insoluble fractions of B. burgdorferi, suggesting association with both inner and outer bacterial cell membranes. Treatment of intact B. burgdorferi with proteinase K partially digested BmpA, consistent with a limited surface exposure on the outer bacterial membrane, a suggestion confirmed by immunofluorescence of unfixed B. burgdorferi cultured in vitro and in vivo. Anti-rBmpA antibody was bacteriostatic for B. burgdorferi B31 in culture, again suggesting localization of BmpA on the exposed spirochetal outer surface. Surface localization of BmpA, growth inhibition by anti-rBmpA antibodies, and the previously reported conservation of bmpA in different B. burgdorferi sensu lato strains may indicate that BmpA plays an essential role in B. burgdorferi biology.