Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-I virus were synthesized. Monomeric peptides P7 and PS represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P7 is a gp21 (374-400) sequence and the peptide PS is a gp46 (190-207) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P7-GG-P8 and P8-GG-P7), separated by two glycine residues as spacer arms. The antigenic activity of these peptides were evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-1-positive sera (n = 22), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-II-positive sera (n = 11), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and monomeric peptides together. The chimeric peptide P7-GG-P8 proved to be the most reactive with anti-HTLV-1-positive sera. These results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-1 diagnostics.