In this study, we characterized a novel Babesia bovis cDNA clone obtained by immunoscreening the cDNA expression phage library with B. bovis-infected bovine serum. The genetic analyses showed that it contained an open reading frame of 993 bp, which was considered to encode B. bovis L-lactate dehydrogenase (BbLDH: E.C. 1.1.1.27) because of the strikingly high amino acid identities of its gene product to the LDHs of Plasmodium falciparum and Toxoplasma gondii. Immunological analyses with the anti-recombinant BbLDH mouse serum showed that 36 kDa of the native BbLDH was expressed not only in the cytoplasm of intra- and extraerythrocytic parasites but also along the membrane of infected erythrocytes. The kinetic properties of recombinant BbLDH proved a certain enzymatic activity of LDH, and the activity was significantly inhibited by the addition of gossypol, a competitive inhibitor of protozoan LDHs. Moreover, 100 microM of the gossypol irretrievably arrested the in vitro growth of B. bovis. The results demonstrated that BbLDH provides a suitable drug target for the design of novel babesial chemotherapeutics.