&NA;A 23‐amino‐acid‐long peptide (AIEKYLEDQAQLNAWGCAFRQVC) representing the transmembranous protein gp32 in SIVmacwas used in site‐directed enzyme‐linked immunosorbent assay (ELISA) for detection of HIV‐2‐specific antibodies in 567 sera from Bissau, Guinea Bissau. Ninety out of the 567 sera were identified to contain HIV‐2 antibodies by whole antigen ELISA and Western blot assays. The peptide ELISA correctly identified 89 out of these 90 seropositives (sensitivity 98.9%). Three sera falsely interpreted to be positive were encountered (specificity 99.4%). The HIV‐2 peptide was also used for testing of 93 HIV‐1‐positive Swedish sera. None of these sera reacted. Site‐directed serology employing synthetic peptides should be considered for application as a screening assay.