Antibodies to Ro60KD protein are found with high frequency in sera from patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS). Two major epitopes of the Ro60KD antigen, the TKYKQRNGWSHKDLLRSHLKP (169-190) and the ELYKEKALSVETEKLLKYLEAV (211-232), were synthesized and their antigenic and structural properties were studied. Using a large panel of SLE and pSS patients' sera, it was found that the anti-Ro60KD reactivity of both Ro60KD epitopes is rather limited (≈45%), although they retain their original disease specificity. The epitope p. 169-190 possessed sequence similarity with the peptide RPDAEYWNSQKDLLEQKRGR, shared in the β-chain of different HLA-DR molecules, among them the HLA-DR3 (which is associated with anti-Ro/Sjögren's syndrome A (SSA) response in patients with SLE). The antigenicity of the HLA-DR3 RPDAEYWNSQKDLLEQKRGR peptide was found to be similar to the 169-190 homologous Ro60KD epitope, recognized mainly by SLE sera. Structural studies showed that the 211-232 Ro60KD epitope exhibits pronounced helical characteristics, while the 169-190 epitope and the HLA-DR3 homologous peptide possess a somewhat lower percentage of α-helix. A β-folded structure was identified in the latter two peptides. Although the diagnostic value of the reported Ro60KD epitopes seems to be rather limited, correlations with other ribonucleoprotein epitopes (La/Sjögren's syndrome B, Ro52KD) may prove complementary to each other and valuable in clinical use. The ordered structure of the HLA-DR3 homologous peptide, exposed to the autoantibody binding, may offer an initiative in further investigation of the role of the HLA haplotypes, associated with the anti-Ro/SSA response, in the autoimmune stimulus.