A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B. gibsoni-infected dog serum. cDNA encoding 29-kDa protein was cloned and designated as the P29 gene. The complete nucleotide sequence of the P29 gene was 792 bp. Computer analysis suggested that the sequence of the P29 gene contained an open reading frame of 597 bp with a coding capacity of approximately 23.4 kDa and a single intron of 250 bp. The P29 protein had homology to Toxoplasma gondii cytoskeletal protein IMC1. Southern blot analysis indicated that the P29 gene was present as a single copy in the B. gibsoni genome. The native P29 protein of B. gibsoni with a molecular mass of 29 kDa was identified by Western blotting with anti-recombinant P29 mouse serum. Confocal laser microscopic analysis showed that the P29 protein was located on the cytoplasma of B. gibsoni merozoites. The recombinant P29 protein expressed in E. coli was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis subspecies-infected dog serum or normal dog serum. Furthermore, the antibody response against the P29 protein was maintained during the chronic stage of infection in an experimentally infected dog, indicating that the recombinant P29 protein might be a useful diagnostic reagent for the detection of antibodies to B. gibsoni in dogs.