A panel of high- and low-titre anti-RNP-positive patient sera was tested for reactivity with human snRNP proteins. The U1 snRNP-specific 70-kD peptide was found to be the most prominent RNP antigen recognized by high-titre anti-RNP sera, mainly found in patients with mixed connective tissue disease (MCTD). The reactivity with the 70-kD protein was further analysed with recombinant fusion proteins containing different segments of the protein. One major and two minor antigenic regions were found. Most patient sera, both with high- and low-titre anti-RNP antibodies, only recognized the major region. The most aminoterminal region, showing partial sequence homology with a mouse retroviral p30 gag protein, contained an epitope that was recognized by one serum only.