The first part of this study of the biology Cal mechanisms underlying attenuation of virulent Theileria annulata macroschizont-infected cell Lines screened four pairs of T. annulata (Hisar) in vivo- and in vitro-derived macroschizont-infected cell Lines (lines) and identified a single in vivo-derived line, which induced lethal tropical theileriosis. The other seven lines were relatively avirulent. Analysis of the clinical, hematological, and parasitological responses of cattle immunized with different passages of the virulent line after in vitro culture showed that it was partly attenuated by passage (p) 50 and avirulent by p130. Clones representing the three glucose phosphate isomerase (GPI) isotypes, which constituted the newly isolated virulent culture, were obtained from p3 by limiting dilution: p50 and p130 consisted of one isotype. The second part of the study raised monoclonal antibodies (MAbs) against macroschizont-infected cells, as reagents for detecting antigenic differences between virulent and avirulent parasites, and identified two MAbs that recognized the surface of infected cells as well as macroschizonts, MAb EU1 recognized an antigen expressed by all the lines tested, whether in vitro- or in vivo-derived, whether uncloned or cloned, and irrespective of extent of subpassage in culture. MAb EU106 recognized an antigen whose expression by the virulent line and its clones disappeared on passage in culture. This antigen was not expressed at all by the avirulent iir vitro-derived line prepared with cells from the same calf Both antigens were expressed by lines infected with other stocks of T. annulata, including two lines known to induce lethal disease. The different profiles of expression of the two novel antigens, recognized by MAbs EU1 and EU106, by the line undergoing attenuation suggest (1) that the two antigens interact differently with the bovine immune system; and (2) that there are two, very different, potential roles for these antibodies in the development of vaccines against T. annulata infections.