One hundred and fifty-one human sera from patients exposed to rubella virus (RV) and shown to be negative for IgM antibodies were tested for total RV-IgG, hemagglutination inhibition (HAI) and for virus neutralizing (VN) antibodies using a peptide enzyme-linked immunosorbent assay (ELISA) based on BCH-178, a peptide representing one of several known neutralizing epitopes on RV hemagglutinin (E1). The data showed that, among 39 and 51 sera with HAI and RV-IgG titres of 1/128 and >150 IU/ml, respectively, neutralizing antibody readings using the BCH-178 ELISA were above cut-off values. However, 13% of HAI positive sera (titre > or =1/16) and 16% of RV-IgG ELISA positive sera (> or =20 IU/ml) were below the cut-off value of the BCH-178 ELISA. This may explain why several cases of congenital rubella syndrome (CRS) have been observed in spite of positive titres. We suggest that a diagnosis of sufficient immunity against RV infection or reinfection may be safer if an additional test detecting antibodies against VN RV epitopes is positive as well.