Attenuated Babesia bigemina were multiplied in vivo and in vitro to vaccinate two groups (Groups 2 and 3) of Holstein Friesian heifers. Another group (Group 1) of heifers was vaccinated twice with purified soluble antigens obtained from the supernatant of in vitro culture combined with saponin. All these heifers plus controls (Group 4) were inoculated with heterologous pathogenic B. bigemina 5 months later. Heifers vaccinated with live organisms (Group 2 and 3) were able to stand the challenge without specific treatment whereas the opposite occurred in heifers vaccinated with soluble antigens (Group 1) and controls (Group 4). Antibody titres were higher in heifers inoculated with soluble antigens than in heifers inoculated with live B. bigemina multiplied in vivo, indicating that antibody titres may not be a proper indicator of protection. In vitro culture of this protozoan is probably a better source of live antigens for vaccine production than in vivo culture. These kind of immunogens can fill the gap until improved vaccines are available.