Technical Abstract: Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4+ T lymphocytes and IFN-? production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4+ T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were previously shown to stimulate memory CD4+ lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20 kDa CFE fraction identified a 20 kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20 kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions previously shown to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4+ T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4+ T cell clones proliferated in response to several B. bovis strains and produced IFN-?. BLAST analysis revealed significant similarity of the Bbo20 a crystallin family