Peripheral blood mononuclear cells from a large number of human immunodeficiency virus (HIV)-seropositive donors were used to analyze the CD8+ T-cell response to each part of the Nef protein of HIV-1/LAI. This report identifies an immunodominant region (amino acids 73 to 144) in the Nef protein that was recognized by 97% of the NEF responder donors. This peptide sequence was dissected into four epitopic regions (amino acids 73 to 82, 83 to 97, 113 to 128, and 126 to 144), each of which was recognized under different HLA class I restrictions. Short overlapping peptides were used to sensitive the target cells for cytolysis and so to determine if these epitopic regions were multirestricted. Each region was found to contain several epitopes recognized with different HLA molecules. Thus, the central region of the Nef protein, a regulatory protein expressed early in HIV-infected cells, is rich in epitopic sequences which are found to be similar in many infected individuals and which can be recognized in association with at least ten HLA class I molecules. Their implications for the vaccination of humans with peptide sequences are discussed.