Serological comparisons among piroplasm antigens of the benign Theileria species of cattle from Japan, Australia and Britain, which are frequently referred to as T. sergenti, T. buffeli and T. orientalis, were carried out. The results obtained from comparative enzyme-linked immunosorbent assay (ELISA) using sera from infected cattle suggest that T. sergenti could be differentiated from both T. buffeli and T. orientalis by their serological dissimilarities. Western blotting combined with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) revealed that both the 33-kDa piroplasm protein of T. sergenti and the similar 34-kDa protein of T. buffeli and T. orientalis corresponded to immunodominant antigens against cattle. The other 32-kDa proteins of T. buffeli and T. orientalis also represented immunodominant antigens. Cross-reactivities of the 32- and 34-kDa proteins were observed between T. buffeli and T. orientalis, whereas the 33-kDa protein of T. sergenti could be differentiated from the similar 34-kDa proteins of T. buffeli/orientalis. The present study suggests that T. sergenti should be separated from T. buffeli and T. orientalis on the basis of their serological dissimilarities.