Abstract: Highly immunoreactive regions of gp41 of HIV-1, gp32 of HIV-2 and p24 of HIV-1 were identified using synthetic peptides. Superior immunoassay performance is obtained with these peptides linked to carrier proteins as compared to use of the free peptides. Additional natural and unnatural variants of these reactive regions to define a set of peptides that, as cysteine-linked peptide-protein conjugates, provide optimal immunoassay performance including high immunoreactivity with HIV antibody positive samples, low reactivity with negative samples, high discrimination between positives and negatives, and high specificity. These peptide conjugates further permit simultaneous detection of HIV-1 and HIV-2 antibodies, and make possible rapid and simple test formats that require no instrumentation for detection of these antibodies.