High-titre equine immune sera were used to screen a lambda gt 11 expression library of Babesia equi cDNA fragments. Two cDNA clones which did not cross-hybridize to each other were studied. Both clones hybridized specifically to DNA from B. equi but not to DNA from B. caballi, B. divergens or B. ovis. Recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins with apparent molecular weights of 40 kDa and 75 kDa. Polyclonal antibodies directed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa respectively in crude lysates of B. equi merozoites indicating that neither cDNA clone was full length (GST = 26 kDa). In Western blotting experiments the 75 kDa protein showed cross-reactivity with sera from horses infected with B. caballi and was not further investigated. The 40 kDa protein was additionally tested in an enzyme-linked immunosorbent assay (ELISA). A test was developed which had a calculated specificity of 99% and a sensitivity of 88% with sera from horses infected with the homologous strain of B. equi. The ELISA did not recognize sera from horses infected with B. equi strains from Brazil and Morocco.