A strategy for the stable expression of proteins, or large protein fragments, from Chlamydia trachomatis into human cells was designed to identify bacterial epitopes endogenously processed and presented by HLA-B27. Fusion protein constructs in which the green fluorescence protein gene was placed at the 5’-end of the bacterial DNA primase gene or some of its fragments were transfected into B*2705-C1R cells. One of these constructs, including residues 90-450 of the bacterial protein was stably and efficiently expressed. Mass spectrometry-based comparative analysis of HLA-B27-bound peptide pools lead to identification of 3 HLA-B27 ligands differentially presented in the transfectant cells. Sequencing of these peptides confirmed that they derived from the bacterial DNA primase. One of them, spanning residues 211-221, showed 55% sequence identity with a known self-ligand of HLA-B27 derived from its own molecule. The other two bacterial ligands, P(112-121) and P(112-122) derived from the same region and differed in length by one residue at the C-terminus. Both peptides showed >50% identity with multiple human protein sequences that possessed the optimal peptide motifs for HLA-B27 binding. Thus, expression of proteins from arthritogenic bacteria in HLA-B27-positive human cells allows identifying bacterial peptides that are endogenously processed and presented by HLA-B27 and show molecular mimicry with known self-ligands of this molecule and human proteins.