The immunodominant regions of the gp41 from 13 HIV-1 subtype O strains from Cameroon, 11 from France and one from Germany were sequenced. The amino acid sequences were compared to those of the 3 published HIV-1 subtype O isolates, ANT70, MVP-5180 and VAU. All HIV-1 subtype O isolates had a very conserved amino acid sequence in this region and showed a subtype O specific structure. Within the cysteine loop there was a positive charge of two basic amino acids, arginine and lysine. Only two strains (CM.6778 and CM.8161) showed an acidic amino acid in this loop. None of the isolates showed the same amino acid sequence in this immunodominant region. A 25 residue peptide from the immunodominant domain of gp41 of the MVP-5180 strain was synthesized, cycled to form the cysteine-loop and coated to microtiter plates. Antibody binding was detected by indirect ELISA using an enzyme labeled anti-human IgG. Out of 111 anti-HIV-1 positive specimens, collected mainly from Cameroonian HIV infected patients, only 10 were not reactive in this assay. The 42 anti-HIV-1 subtype O positive specimens gave all a reaction above cut off. Despite the diversity found in the amino acid sequences within the 25 isolates a peptide-based indirect ELISA representing the immunodominant epitope of the strain MVP-5180 successfully detected all the anti-HIV-O sera so far tested, pointing to the importance of adding such a peptide for correct identification of HIV-1 subtype O infected patients, while some assays without HIV-O specific antigens partially fail to detect all anti-HIV-O specimens.