Mice immunized with plasmid DNA encoding Nef regulatory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies. After 4 intramuscular injections of 100 microg plasmid DNA, anti-Nef antibodies reached titers up to 2 x 10(4). A significant specific antibody response was maintained for at least 16 months. Using a set of seven 31-66 mer synthetic peptides covering the entire sequence of Nef, we analysed the specificity of ant-Nef antibodies. Interestingly, specific antibodies produced in response to Nef expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide (aa 141-205) for 3 of the 10 sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. Only 3 of the 5 Nef positive sera reacted with the C-terminal peptide. This suggests that specific antibodies induced by plasmid DNA as well as by the non-denatured protein recognize conformation-dependent epitopes. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.