Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and ofnef‐protein from HIV‐1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4‐(oxymethyl)‐phenylacetamidomethyl resin using the N‐α‐butyloxycarbonyl amino acids with benzyl‐based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc‐amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc‐amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non‐polar (TFE) environments. Immunoreactivity of anti‐nefpositive sera from HIV‐1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions ofnef‐protein and mainly the immunodominance of theN‐andC‐termini of the molecule. Several of these peptides should prove to be useful for both diag