Ovine babesiosis, caused by the intra-erythrocytic protozoan parasite Babesia ovis, is an infectious and economically important tick-borne disease of sheep. Diagnostic testing is an essential tool used for the control of the disease. In order to identify and characterize the immunoreactive proteins which are useful in serological diagnosis of the disease, a complementary DNA (cDNA) expression library was constructed from B. ovis merozoite mRNA. A cDNA clone designated as BoSA2 was identified by immunoscreening of a cDNA library using immune sheep serum. The sequence of the BoSA2 cDNA had a partial open reading frame of 1156 nucleotides encoding a polypeptide of 384 amino acid residues. Theoretical molecular mass for the mature protein was 43.5. kDa. The sequence of the BoSA2 was inserted into the expression vector pGEX-4T-1 and then expressed in Escherichia coli DH5α cells as a glutathione S-transferase (GST)-tagged fusion protein. This recombinant fusion protein (rBoSA2) was purified by GST-affinity chromatography. Immunoreactivity of the rBoSA2 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using the sera from the animals naturally and experimentally infected with B. ovis. ELISA results demonstrated that this antigen was useful for the diagnosis of ovine babesiosis. The localization of the BoSA2 protein was shown in and on the parasite and in the cytoplasm of the infected erythrocyte by confocal laser microscope. To our knowledge, rBoSA2 is the second immunoreactive recombinant protein of B. ovis until the present.