Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. 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.