It was recently shown that antibodies reactive with a peptide from the tip of the HIV-1NY5 gp120 V3 loop (V3 peptide) are present not only in sera of HIV-positive patients but also in sera of healthy HIV-negative individuals. In the present study, we show that V3 peptide reactive antibodies are predominantly IgM in sera of HIV negative individuals and that a fraction of the IgG anti-V3 antibodies exhibit features of autoantibodies. These antibodies were purified by chromatography on IgG-sepharose columns from sera as well as from purified IgG anti-V3 antibodies. A higher IgG anti-V3 reactivity was detected in autoantibody preparations from HIV-positive sera as compared with the reactivity of sera and purified antibodies from HIV-negative individuals. This was confirmed by solid phase binding of IgG anti-V3 antibodies both to V3 and to human IgG F(ab')2 antigens. The autoantibodies did not bind to peptides that share sequence similarity with V3 peptide indicating a high epitope specificity. The detection of antibodies against HIV epitopes in HIV-negative individuals may suggest that anti-V3 antibodies after HIV infection represent at least in part a secondary immune response.