Murine monoclonal antibodies (MAbs) raised against a recombinant nef protein fragment of human immunodeficiency virus type 1 (HIV-1) strain BH10 were characterized by an epitope mapping system using overlapping decapeptides. Four different immunogenic regions were identified. Ten human HIV-1-positive sera were tested in the same epitope mapping system, seven of these were reactive with four immunogenic regions. Two of the nef-specific epitopes recognized by human sera overlapped with the epitopes defined by the murine monoclonal antibodies. The reactivity of the monoclonal antibodies with the recombinant nef protein and with infected and uninfected cells were investigated in a variety of test systems. The results are discussed with respect to homologous regions of nef and cellular proteins.