Abstract: Synthetic peptides have frequently replaced the more costly recombinant proteins or viral lysates as the antigens of choice for detection of antibodies to human immunodeficiency viruses. However, development of an assay that is sensitive to all the types and groups of HIV, including the divergent strains of HIV-1 group O, group N, and HIV-2, would require many peptides derived from different types and groups of HIV. Combining multiple peptide antigens may reduce the analytical sensitivity of the individual peptide due to the competition for binding to the solid surface when used in an enzyme immunoassay format. In this study, we developed and evaluated two chimeric multiple antigenic peptides (CMAP) for simultaneous detection of specific antibodies to HIV-1 groups M, N, O, and HIV-2. Both CMAPs correctly identified 304 known HIV positive serum or plasma specimens (260 HIV-1 group M of varying subtypes, 3 group O, and 41 HIV-2) and one chimpanzee serum specimen (group N) and all 66 known HIV negative specimens. CMAP performance was superior to the corresponding individual linear peptides or a linear peptide mixture. The results indicate that CMAPs are useful for the development of highly sensitive and specific assays for the detection of infections caused by HIV-1, including group M, N, and O, and HIV-2. [Copyright &y& Elsevier]