Enzyme-linked immunosorbent assays (ELISA) and immunoblots using either whole-cell lysates of Borrelia burgdorferi or an antigenic region of flagellin (41-G) as the antigen were performed, and the abilities of the two assays to detect antibodies to this spirochete in dog, cottontail rabbit, and horse sera were compared. Assays using whole-cell B. burgdorferi lysates as the antigen were more sensitive for detecting antibodies. ELISA with 41-G as the antigen were specific for Borrelia antibodies but were not as sensitive as the assays with whole-cell lysates coated to the solid phase. Use of recombinant full-length flagellin, rather than 41-G, as the antigen in immunoblots increased the sensitivity of each assay. However, antibodies to other bacterial antigens cross-react with whole flagellin and may account for false-positive results. Antibodies to B. burgdorferi outer surface protein A or B were usually undetected when the sera were tested by immunoblotting methods. Borrelia lysates or the 41-G antigen may be used in ELISA or immunoblots to document host exposure to this spirochete. The use of 41-G as the antigen may increase the specificity of an assay or help confirm the serologic diagnosis of Lyme borreliosis in dogs, horses, and cottontail rabbits.