A serum panel obtained from male homosexuals (n = 278); i.v. drug abusers (n = 99), patients attending a VD clinic (n = 390), blood donors who visited Central or West Africa (n = 573), blood donors who had sexual contact with natives from Central or West Africa (n = 38), blood donors from Surinam, South America (n = 481), individuals positive for anti-HIV-1 (n = 94), individuals with indeterminate HIV-1 western blot (WB) reactions (n = 73), African patients with AIDS or AIDS-related symptoms (n = 30), and random Dutch blood donors (n = 555), was tested with HIV-2 ELISA (ELAVIA-2). Of these 2,611 samples, 32 (1.2%) were repeatedly reactive. Antibodies to gp140/105env were found in 4/32 by HIV-2 WB, and in 1/4 by radioimmunoprecipitation (RIPA). These 4 HIV-2 WB-positive samples were also reactive with gp160/120env in HIV-1 WB, suggesting cross-reactivity. In a spot test with synthetic peptides of the transmembrane glycoprotein of both HIV types, 3/4 were only HIV-1 positive and 1/4 was strongly HIV-2 positive and weakly HIV-1 positive. In inhibition assays with soluble HIV-1 or HIV-2 synthetic peptides in HIV-1 and HIV-2 peptide ELISA, cross-reactivity was excluded, which indicates an HIV variant or HIV-1/HIV-2 double infection. It is concluded that for the moment HIV-2 infection is at low prevalence in risk groups in The Netherlands, and that in addition to WB and RIPA, synthetic peptide assays are useful for differentiation between HIV-1 and HIV-2 antibodies.