BACKGROUND We have recently demonstrated the ability of herpes simplex virus type 1 (HSV-1) to agglutinate mouse red blood cells, and identified glycoprotein C (gC-1) as a major virus hemagglutinin. Based on this a classical hemagglutination-inhibition (HI) assay was developed. OBJECTIVE Regarding significant structural differences between HSV-1 gC-1 and its herpes simplex virus type 2 (HSV-2) counterpart, gC-2, the possibility of application of a classical HI assay for the detection of HSV-1-specific antibodies was explored. METHODS HI antibody titers were compared with those of gC-1-specific enzyme-linked immunoassay (ELISA), and with the results of the standard gG-1- and gG-2-specific immunodot enzymatic assays for the detection of type-specific antibodies to HSV-1 and HSV-2 respectively. RESULTS The sensitivity of HI test was 89% and 97% of that gC-1-ELISA and gG-1-immunodot respectively. Approximately 21% of serum specimens, defined as containing antibodies specific for only HSV-2, showed low HI titers. Heterotypic reactivity with purified gC-1 antigen was also observed in both ELISA and immunoblot assays. CONCLUSIONS Antibodies detectable in HI assay were mainly HSV-1-specific; however, a limited degree of serologic reactivity between HSV-2-specific sera and HSV-1 hemagglutinin also occurred. Thus, our results confirmed prevalent opinion about the presence of a limited number of antigenic determinants shared by HSV-1 gC-1 and HSV-2 gC-2.