The hemoparasite Babesia bovis antigenically alters the bovine erythrocyte membrane surface by expression of isolate-specific, parasite-derived polypeptides. To determine whether antigenic variation also occurred on the infected erythrocyte surface, a calf was infected once with parasitized erythrocytes carrying the C9.1 clonal line of B. bovis. In vitro cultures then were established periodically from the peripheral blood and analyzed with sequentially collected sera from the same animal. The surface reactivity of infected erythrocytes cultured from the infected animal varied over time, on the basis of reactivity in live cell immunofluorescence, surface immunoprecipitation, and panning assays. Subclones C8 and H10, established from day 41 cultures, were analyzed immunochemically. A loss of immunoreactivity was observed in antigens corresponding to the 113- and 128-kDa parasite-derived antigens of clone C9.1, demonstrating epitopic variation in these antigens; the immunochemical recognition of these antigens paralleled the results of live cell immunofluorescence and panning assays. Concomitant size polymorphism suggested polypeptide structural variation of these antigens as well. Calves infected by inoculation of infected blood or by injection of cloned parasites from in vitro cultures rapidly developed antibodies which cross-reacted among the clonal variant lines, suggesting the presence of common as well as unique epitopes. These results demonstrate that antigenic variation occurs on the surface of B. bovis-infected erythrocytes and that the parasite-derived antigens of 113 and 128 kDa compose at least a part of the antigens undergoing variation.