The nuclear lamina of mammalian cells consists of three major proteins, lamins A, B and C, which form a fibrous meshwork interposed between the inner nuclear membrane and the chromatin. Sera from certain patients with systemic lupus erythematosus (SLE) and autoimmune liver disease contain high titers of autoantibodies against lamin B. We have shown previously that anti-lamin B autoantibodies in SLE recognize epitopes highly specific for lamin B, even though lamin B and lamins A/C are highly homologous proteins. To further characterize the specificities of these autoantibodies, fusion proteins carrying fragments of lamins B and C were tested for reactivity with SLE sera by immunoblotting. Five distinct epitopes of lamin B were identified, at least four of which were located in the highly conserved coiled-coil rod domain. Epitopes located on amino acids (AA) 80-193 and 245-303 were recognized by 4/10 and 8/10 anti-lamin B positive sera, respectively. Affinity purified anti-lamin B autoantibodies reacted preferentially with lamin B, indicating that they recognized mainly portions of lamin B that differ from lamins A and C. On the contrary, most of the affinity-purified anti-lamin C autoantibodies from SLE sera cross-reacted with lamin B, suggesting that the anti-nuclear lamina immune response in these patients is directed primarily against lamin B. The preferential reactivity of these sera with multiple epitopes specific to lamin B, and the finding that the autoantibodies to lamins A and C present in some of these sera cross-react with lamin B suggest that autoantibodies to lamin B are generated in response to the authentic lamin B protein rather than a cross-reactive foreign protein.