The efficacy of the Be82 gene product fused with glutathione S-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Babesia equi infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST/Be82 antigen cross-reacted with Babesia caballi-infected horse sera, despite the high rate of detection of B. equi. These results suggested that GST/Be82 has an antigen in common with B. caballi or antigenicity similar to that of B. caballi. In the present study, we constructed a series of five clones with deletions in the Be82 gene product, each of which was fused with GST, and used them in ELISAs in order to overcome the cross-reactivity seen with B. caballi. One of the deletion clones, a clone with a deletion of the Be82 gene from positions 236 to 381 (Be82/236-381), specifically and sensitively detected B. equi-infected horse sera without cross-reactivity with B. caballi-infected horse sera. Assays with clones from which other gene products were deleted showed decreased sensitivities or remained nonspecific for the detection of B. equi-infected horse sera. These results suggest that the Be82/236-381 gene product is a novel antigen for the diagnosis of B. equi infection in horses.