HLA-B27-related arthritis is probably mediated by an immune response against HLA-B27 complexed with peptides derived from proteins of arthritis-causing bacteria. Immunogenic proteins with a high degree of homology among bacteria, such as in the hsp60 family, are likely candidates. To create such complexes experimentally, we transfected an HLA-B27 cell line with the Mycobacterium tuberculosis hsp60 gene. Because of previous observations that HLA-B27-peptide complexes can be distinguished by antibodies, we tested the transfected cell line with a panel of sera from 24 HLA-B27+ arthritis patients. Significant antibodies were detected in at least eight of the sera. Several cell lines and peptides were used as negative controls to ensure that the antibody reactivity was specific to HLA-B27-peptide complexes. A panel of nine peptides derived from the sequence of the Mycobacterium hsp60 were synthesized and tested. At least three were identified as being responsible for the serological activities.