Antigen-specific CD4(+) T-lymphocyte responses are restricted by major histocompatibility complex class II molecules, which influence T-cell priming during infection. Human leukocyte antigen (HLA) and bovine leukocyte antigen (BoLA) DRB3 and DQ genes are polymorphic, but unlike HLA, many BoLA haplotypes have duplicated DQ genes, and antibody-blocking studies indicated that BoLA-DQ molecules present various pathogen epitopes. Limited experimentation also suggested that BoLA-DQ molecules formed by interhaplotype pairing of A and B chains are functional. To compare antigen presentation by DR and DQ molecules and to definitively demonstrate functional BoLA-DQ molecules derived from interhaplotype pairing, different combinations of DR or DQ A and B proteins were expressed with CD80 in 293-F cells for use as antigen-presenting cells (APCs). This approach identified 11 unique restriction elements including five DR and six DQ pairs for antigen-specific CD4(+) T-cell responses against tick-transmitted bovine hemoparasites Anaplasma marginale or Babesia bovis. Interhaplotype pairing of DQ A and B molecules was demonstrated. Testing of six expressed DQA/B pairs from an animal with duplicated DQ haplotypes (DH16A/DH22H) demonstrated that an interhaplotype pair, DQA*2206/DQB*1301, presented A. marginale peptide B. In DH22H and DH16A homozygous animals, DQA*2206 was tightly linked with DQB*1402, and DQA*22021 was linked with DQB*1301. APCs from these donors could not present peptide B, confirming that DQA*2206/DQB*1301 encoded a functional interhaplotype pair. Functional BoLA-DQ molecules are generated by both intrahaplotype and interhaplotype pairing of A and B chains and play a similar role to BoLA-DR in priming helper T-cell responses to important pathogens.