The reason for the high association of HLA-B27 with diseases such as ankylosing spondylitis and reactive arthritis is not clear. In reactive arthritis, the triggering bacteria are known, thus allowing investigation of their interaction with HLA-B27. CTL lines derived from five patients with Yersinia-induced reactive arthritis were raised by repeated stimulation in vitro with either Yersinia-infected autologous macrophages (four patients) or pooled peptides (three patients) having the HLA-B27-binding motif. The peptides were derived from five Yersinia proteins and from the chlamydial 57-kDa heat shock protein (hsp). Cytotoxicity of T cell lines was then tested against these peptides. Lytic activity was obtained with T cells stimulated with viable Yersinia or pooled peptides. Targets successfully used for lysis were cells pulsed with peptides from the Yersinia 60-kDa hsp, but not cells pulsed with peptides from other Yersinia proteins or the chlamydial hsp. T cell lines raised with 60-kDa peptides also lysed targets infected with Yersinia. Most interestingly, all three CTL lines tested (one raised with Yersinia; two with pool of peptides) recognized only one single peptide (321-329) of seven tested from the Yersinia hsp60. Cytotoxicity occurred only when target cells were matched for HLA-B27. This identification of an immunogenic peptide derived from an arthritogenic bacterium and presented by HLA-B27 opens the way for future investigation of the role of T cells specific for this peptide or cross-reacting peptides, in the immunopathology of HLA-B27-associated diseases.