Herpes simplex virus type 1 (HSV-1) has been recently known to agglutinate mouse erythrocytes. We performed the hemagglutination (HA) test under various conditions to investigate the nature of hemagglutinin of HSV-1. HA of mouse erythrocytes was observed with glycoprotein C (gC)-positive HSV-1 strains but not with gC-negative HSV-1 mutants and HSV-2 strains. A recombinant HSV-1 constructed by transfecting a gC-negative strain with a gC-1 gene regained full HA activity. The erythrocytes from several strains of rats as well as mice were agglutinated by gC-positive HSV-1 strains. Formalin fixation of erythrocytes and ultraviolet inactivation of virus did not reduce the HA activity. The HA activity which was inhibited by heparin and dextran sulfate appeared late in the process of virus multiplication and was inhibited by adding cytosine arabinoside or tunicamycin to the medium. The HA activity was inhibited not only by antisera against HSV-1 but by antisera against HSV-2, although their hemagglutination inhibition antibody titers were low. These date support the importance of gC in HSV-1 and heparan sulfate moieties at the surface of erythrocytes as previously reported. HA by HSV-1 might provide a new serological diagnostic test for HSV infection.