Potential relationships between amino acid motifs in the antigen binding groove of various alleles of the bovine major histocompatibility complex DR (BoLA-DR) molecule and Peripheral Blood Mononuclear Cell (PBMC) proliferation, in response to S. aureus and phytohemaglutinin, were investigated. The animals included in the study were approximately of the same age and comprised of pure Holstein-Friesian (n = 86), F2 Holstein-Friesian Charolais (n = 155), Holstein-Friesian backcross (F0 Holstein-Friesian dams crossed with unrelated F1 sires, n = 60), Charolais backcross (F1 dams crossed with F0Charolais sire, n = 46). To determine BoLA-DRB3 alleles of the animals, a sequence-based typing method was employed. The amino acid sequence data encode various BoLA-DRB3 alleles, presented at the latest BoLA workshop, were utilized to determine the residues involved in the formation of the pockets in the antigen binding groove of various alleles of the BoLA-DR molecules. A linear mixed model was utilized to evaluate the potential relationships between amino acid motifs in the antigen-binding groove of various alleles of BoLA-DR molecule and PBMC proliferation.