The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni–infected, Babesia canis–infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coli is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.