A cDNA encoding the Babesia microti 32-kDa protein was identified by serological immunoscreening of a cDNA expression library and designated as BmP32. The full length of BmP32 contains an open reading frame of 918 base pairs consisting of 306 amino acids having a significant homology with B. microti secreted antigen 1. Antiserum raised against recombinant protein (rBmP32) specifically reacted with a 32-kDa native protein of the parasite lysate using western blot analysis. The indirect immunofluorescent antibody test showed a preferable localization of BmP32 in the cytoplasm of the intra- and extracellular parasites. Moreover, BmP32 was secreted in the cytosol of infected erythrocytes, especially during the peak parasitemia and the recovery phase of the infection. Next, the antigenicity of rBmP32 was examined by an enzyme-linked immunosorbent assay (ELISA) and sera from mice experimentally infected with either B. microti or closely related parasites. ELISA was highly specific and sensitive when used for the detection of B. microti antibody in a mouse model. Furthermore, mice immunized with rBmP32 emulsified with Freund's adjuvant were not significantly protected against challenge infection with B. microti. However, high antibody titer was detected just before the challenge infection. Our data suggest that rBmP32 may be a specific diagnostic antigen but not a subunit vaccine.