One of the most common cattle major histocompatibility complex DRB3 alleles, *0201, includes a deletion of codon 65 encoding one residue in the alpha-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, *3301 and *4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11-17 nucleotide substitutions causing 8-11 amino acid substitutions) we found clear indications of a common ancestry. The alpha-helical region was very similar or identical among the alleles whereas the beta-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles.