This article reports the HIV epitope specificity of antibodies present in the sera of HIV-negative patients with autoimmune diseases. Recombinant gp120 and a panel of synthetic peptides derived from the amino acid consensus sequences of either related (gp120, gp41, and p24) or unrelated (Mage-1, necdin, heat shock protein [65 kDa], and amyloid) HIV proteins were tested by a specific ELISA. The first set of experiments performed on four patients with Sjögren's syndrome (SjS) and four patients with systemic lupus erythematosus (SLE) revealed a significant anti-gp120 antibody reactivity in autoimmune patients when compared to healthy HIV-negative controls. Moreover, such binding could be almost completely inhibited by preincubation with free gp120. A significant anti-p24 reactivity was observed in 18 of 29 sera from SjS patients and in 13 of 25 sera from SLE patients, while anti-gp41 was observed only in 3 of 14 SjS and in 2 of 20 SLE-affected patients. Similar analyses were performed in the murine model of autoimmunity, showing that sera from MRL/lpr mice were able to bind all HIV-related peptides in an age-dependent manner. The analysis of a panel of HIV-unrelated peptides showed that SLE as well as MRL/lpr sera bind both HIV-related and unrelated peptides, while SjS sera failed to do so, revealing the polyclonal nature of the SLE and MRL/lpr repertoire and the oligoclonal reactivity of SjS sera. This is also supported by inhibition experiments, which showed that SLE, but not SjS, sera competitively inhibited the binding to HIV gp120 peptide of sera from autoimmune MRL/lpr mice. These results indicate that an overlapping polyclonal repertoire is present in both SLE and MRL/lpr sera, while the oligoclonal specificity of SjS antibodies may be related to a specific, nonpolyclonal, activation against putative retroviral antigens.