A peptide of 21 amino acids with the sequence Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-GlnLeu-Leu-Gly-Ile-Trp-Gly-Cys-Ser encoded by a segment in the env gene of human T-lymphotropic virus type m (HTLV-Il), corresponding to amino acids 586-606 of the precursor envelope glycoprotein, has been synthesized by the Merrifield solid-phase method. Combined serological and chemical analyses of this peptide and related peptides have revealed the importance of certain amino acid residues in the antigenic determinant of the relevant peptide. Enzyme immunoassay (EIA) employing this peptide as an antigen adsorbent was shown to reproducibly detect antibodies in sera of patients with HTLV-II infection. This assay provided positive results with all sera that were reactive with gp4l envelope protein of HTLV-Ill in electrophoretic immunoblot analysis. Thus far, no false-positive sera have been encountered in control populations. Our EIA with this peptide as the coating antigen is shown to have advantages over that with the whole HTLV-llI virus as an immunoadsorbent.Since the identification of a human retrovirus, named human T-lymphotropic virus type III (HTLV-III), acquired immune deficiency syndrome (AIDS)-related virus (ARV), or lymphoadenopathy-associated virus (LAV) as the infectious agent for AIDS (1-4),t substantial progress has been made in the studies of the virus and in the development of diagnostic methods for the detection of HTLV-III infection. With the establishment of permissive T-cell lines for mass production of HTLV-III virus (1), it has been possible to elucidate the structure and gene organization of HTLV-III, to determine the complete nucleotide sequence of the HTLV-III genome (5-8), and to use heat-inactivated HTLV-III as the immunoadsorbent for detection of antibodies against HTLV-III in sera of patients with HTLV-III infection (9)(10)(11)(12)(13)(14)(15)(16)(17)(18).Efforts employing recombinant DNA technology to identify HTLV-III antigenic peptides reactive with sera from AIDS and AIDS-related complex (ARC) patients and asymptomatic HTLV-III-infected individuals have also been undertaken by many investigators (19)(20)(21)(22)(23). Success has been obtained by this approach. However, it has not been able to localize and identify the peptide sequences representing the antigenic epitope(s).Synthetic peptides are used increasingly to map antigenic or immunogenic sites on the surface of proteins and, in turn, as possible vaccines (reviewed in ref. 24). We have synthesized numerous peptides corresponding to various regions of envelope protein of HTLV-III and analyzed their reactivity with sera from AIDS patients. Our initial emphasis was on the gp4l glycoprotein, the transmembrane portion of envelope protein gp160, which has previously been identified as the antigen most consistently recognized by antibodies in patients with AIDS and ARC (25,26).In this paper, we report (i) the identification of a peptide 21 residues in length with the sequence Arg-Ile-Leu-Ala-ValGlu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Le...