Helper T cell clones from two Babesia bovis-immune cattle were characterized for use in identification of potentially protective immunogens of B. bovis merozoites. Proliferation assays with 11 CD4+ clones revealed a differential pattern of response to soluble cytosolic antigen, membrane-enriched antigen, detergent extracts of the membrane-enriched antigen, soluble culture supernatant exoantigen, and different geographical isolates of B. bovis as well as Babesia bigemina parasites. When the data were combined, the clones could be grouped according to five different patterns of response. One group recognized only the membrane-enriched fraction of New World and Australian parasites. Four remaining groups recognized antigens found in the cytosolic as well as the membrane-enriched fraction, and clones representative of each group were used to identify cytosolic antigens fractionated by anion-exchange chromatography with the use of fast-performance liquid chromatography. One clone (C97.3C3), which responded to all B. bovis isolates and to B. bigemina, recognized a single peak of activity that eluted with 0.25 M NaCl and contained protein bands of 70 and 75 kDa. The remaining clones were stimulated by a second antigenic peak that eluted between 0.35 and 0.45 M NaCl and contained protein bands of 42, 47, 56, and 84 kDa. The majority of the clones produced interferon, whereas tumor necrosis factor alpha/tumor necrosis factor beta production was less frequent. These studies provide the basis for using helper T cell clones to identify potentially protective immunogens of B. bovis and delineate a minimum of five helper T cell epitopes recognized by two immune cattle.