Virulent and avirulent K strain (KV and KA) and virulent L strain (LV) Babesia bovis parasites were biosynthetically labeled with 35S-methionine. Labeled proteins, extracted in detergent from infected cultures, were analyzed by two-dimensional gel electrophoresis. Protein antigens common to these forms of babesia and those specific for strain and correlating with the degree of virulence were identified by immunoprecipitation with antisera. Dominant labeled protein antigens were found to differ both between the K and L strains of B. bovis and between virulent (KV) and avirulent (KA) forms of the K strain. Partial cleavage of biosynthetically labeled proteins by staphylococcal V-8 protease and peptide mapping revealed that no extensive amino acid homology existed between the candidate variant antigens. KA B. bovis parasites are currently in use as a live attenuated vaccine for bovine babesiosis in Australia. Antigens that are better expressed in KA relative to virulent strains may therefore be markers of avirulence and/or candidate "host-protective antigens" of B. bovis. At least one protein (Mr 43,000) was identified as being a dominant labeled antigen of KA that is poorly represented in the KV and LV B. bovis isolates.