We mapped an immunodominant domain of the human immunodeficiency virus (HIV). We selected hydrophilic amino acid sequences encoded by conserved regions of the gag, pol, and env genes of HIV as potential antigenic domains. Eighteen peptides representing these sequences were synthesized; the peptides elicited strong antibody responses in rabbits. Sera from 53 HIV-infected patients and 50 controls were tested against the synthetic peptides. Although no antibodies to peptides from gag, pol, or env gp120 proteins were present, antibodies to four of the six peptides from env gp41 were detected. Epitope mapping using overlapping peptides showed that sera from 53 (100%) of 53 HIV-infected patients (and from none of 50 controls) reacted with peptides aa584-609 and aa598-609 from gp41, sera from 32 (60%) of 53 patients reacted with peptide aa603-614, and sera from 19 (35%) of 53 patients reacted with peptides aa609-620. Thus, amino acid sequence LeuGlyIleTrpGlyCysSerGlyLysLeuIleCys (aa598-609) from the transmembrane glycoprotein is an immunodominant domain of HIV recognized by serum antibodies from HIV-infected patients.