HLA-B27 is highly associated with ankylosing spondylitis (AS), but the mechanism is unknown. Among the HLA-B27 alleles, B*2709, which differs by one amino acid from the susceptible B*2705, is not associated with the disease. Here, we analyze the reactivity, in patients with AS and in healthy controls carrying the B*2709 or B*2705 alleles, to an EBV epitope derived from LMP2 (236-244) and to a sequence-related self-peptide from vasoactive intestinal peptide receptor 1 (VIP1R 400-408). We found that both B*2705⁺ and B*2709⁺ subjects possess LMP2 236-244–specific, HLA-B27–restricted T cells, whereas only the B*2705⁺ individuals respond significantly to VIP1R 400-408. These results prompted us to compare, by IFN-γ ELISPOT analysis, the T-cell response to VIP1R 400-408 in patients with AS versus B*2705 healthy controls. The data show that VIP1R 400-408–specific reactivity is a major feature of the patients with AS. These findings show, for the first time to our knowledge, a widespread reactivity in patients with AS against a self-epitope that exhibits some features of a putative “arthritogenic” peptide.