Anti-Ro (or SSA) is found in the sera of patients with autoimmune rheumatic illnesses. All patients with anti-Ro defined by precipitation bind a 60 000 Da antigen (60 kDa Ro), whereas some patients also bind a 52 000 Da molecule (52 kDa Ro). In general, antibody binding is directed against native 60 kDa Ro and denatured 52 kDa Ro. The mechanism by which anti-52 kDa Ro arises in the setting of anti-60 kDa Ro is unknown. Conflicting data exist as to the existence of a physical interaction between the two proteins in cells and as to cross-reacting antibodies. Antibodies were affinity purified from a peptide within the leucine zipper region of 52 kDa Ro. These purified antibodies binding the 197-207 peptide from 52 kDa Ro (anti-52LZ) bound native 60 kDa Ro as well as denatured 52 kDa Ro. In addition, anti-52LZ also bound up to four regions from the sequence of 60 kDa Ro and a single conformational epitope of 60 kDa Ro. Thus, these primary sites represent components of the tertiary epitope. We hypothesized that if this was the case, these peptides making up a tertiary epitope would show molecular interaction. In fact, peptides from 60 kDa Ro have a molecular interaction with the 52 kDa Ro peptide as well as full-length 52 kDa Ro when assessed by surface plasmon resonance. The leucine-zipper region peptide from 52 kDa Ro bound three of the four peptides from 60 kDa Ro. These data suggest that these two molecular species, 60 and 52 kDa Ro, form a conformational epitope. This relationship may explain why anti-52 kDa Ro is found in association with anti-60 kDa Ro.