EcoRI-digested DNA from Borrelia burgdorferi was ligated into the dephosphorylated vector pWR590 and transformed into Escherichia coli DH5α. When the gene library was screened, 20 clones reacted with pooled dog sera with high titers (immunofluorescent antibody titer, ≥1,280) to this spirochete. One clone expressed a 110-kDa antigen that reacted strongly with the high-titered pooled sera from dogs with Lyme borreliosis and serum from goats immunized with B. burgdorferi. The 110-kDa protein was expressed with and without isopropyl-β-D-thiogalactosidase, indicating the protein is not a fusion protein with β-galactosidase. Monospecific antisera to the 110-kDa antigen recognized a 75-kDa Borrelia protein. Of the sera that reacted with B. burgdorferi by immunoblotting; 57, 100, and 83% of human, dog, and horse serum samples, respectively, reacted with the 110-kDa protein. Sera from individuals that tested negative with a B. burgdorferi lysate with immunoblotting showed no reaction with the 110-kDa protein. The 110-kDa antigen appears to be useful for the diagnosis of Lyme borreliosis.