Two chimeric peptides incorporating immunodominant sequences from both HIV-1 (LGIWGCSGKLICTT) and HIV-2 (LNSWGCAFRQVCHT) were synthesized. Peptides KS1-KS2 and KS2-KS1 represented sequences from the two viruses in both possible orders, separated by two glycine residues as spacers. These peptides were evaluated as antigens in an ELISA using a panel of specimens derived from HIV-1 (n = 25) and HIV-2 (n = 25) infected individuals and seronegative people (n = 38). The results were compared to plates coated with individual peptides KS1 and KS2 and to plates coated with two peptides (KS1 and KS2) together. Data demonstrated that individually, KS1 and KS2, are good antigens and can detect antibodies to their respective viruses quite efficiently. However, when coated together, their ability to detect antibodies to both HIV-1 and HIV-2 was reduced, as evidenced by a decrease in OD values obtained. The chimeric peptides KS1-KS2 and KS2-KS1 detected antibodies to HIV-1 and HIV-2; however, their sensitivity of detection was variable and dependent upon the order of their sequence. For both peptides, antibodies directed to the C-terminal portion were detected with higher sensitivity than those directed to the N-terminal part of the peptides. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. Such chimeric antigens may be very useful for simultaneous detection of antibodies to HIV-1 and HIV-2.