Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10(8) cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.