The Sm antigenic complex is, besides dsDNA, the most important and specific autoimmune target in systemic lupus erythematosus (SLE). The population of anti-Sm Ab elicited is very heterogeneous in terms of epitope specificity resulting in a strong assay dependent detectability. Based on the description of a new autoantigenic target, the SmD1-aa83–119 peptide, we analysed 50 healthy persons and 205 patients with different autoimmune and other disorders with regard to their anti-Sm reactivities using different assays. The prevalence of anti-SmD1 peptide Ab and anti-Sm Ab in SLE was 36.0 (40/111) and 9.9% (11/111), respectively. The respective values obtained for non-SLE patients were 2.8 (4/144) and 5.3% (5/94). In SLE, anti-SmD1 peptide Ab are positively correlated to disease activity, nephritis and anti-dsDNA Ab. The association between reactivities of SLE samples in the traditional anti-Sm and the anti-SmD1 peptide ELISA was found to be 63.6%, contrasting markedly with the situation in non-SLE patients (no double-positive sera). SLE samples with an anti-Sm response restricted to the SmD1 peptide are completely negative in immunoblot, supporting the conformational nature of this epitope. Positive immunoblot reactions with the SmD1 polypeptide are not inhabitable by the synthetic SmD1-aa83–119 peptide. Comparing anti-Sm reactivities detected by ELISAs with those in immunoblot, different patterns were observed, reflecting the heterogeneous autoimmune response to this antigen. In conclusion, the anti-SmD1-aa83–119 peptide ELISA substantially completes the panel of methods for autoantibody testing. As none of the assays presently available covers the whole spectrum of epitope specificities of anti-Sm Abs elicited in SLE, it does not replace traditional anti-Sm ELISAs.