We produced recombinant envelope-derived peptides of HIV-2 for use in a diagnostic enzyme-linked immunosorbent assay (ELISA) or Western blot by expressing several restriction enzyme fragments from the env gene of HIV-2 in the bacterial fusion vector pEX-3. On Western blots, 17 out of 18 anti-HIV-2-positive sera available to us reacted strongly with those recombinant peptides which were derived from, or extended into, the transmembrane protein of HIV-2. In contrast, recombinant peptides derived from the external envelope glycoprotein were only weakly recognized by two sera. We observed a cross-reactivity of some human sera containing antibodies to HIV-1 with the HIV-2 peptides derived from the transmembrane protein of HIV-2. In spite of this cross-reactivity, a serological distinction between anti-sera to HIV-1 and HIV-2 can be attempted by simultaneous testing in ELISA on recombinant peptides derived from the transmembrane protein of HIV-1 and HIV-2.