The specificity and neutralizing activity of antibodies against the major herpes simplex virus type 1 (HSV-1) glycoproteins were tested in serum samples of patients with a history of HSV-1 infection. By preabsorption of sera to preparations of native and denatured HSV-1 proteins, followed by immunoblotting and microneutralization, it was shown that the majority of neutralizing antibodies are directed against denaturation-sensitive epitopes. Furthermore, preabsorption of sera to proteins of viral ts and deletion mutants revealed that antibodies specific for gB, gC, and gE had a low neutralizing activity. These results suggest a major role of anti-gD in neutralization of viral infectivity. In addition, it was shown that antibodies directed against the gB monomer were distinct from antibodies against the gB homodimers. The latter, however, did not reveal any measurable neutralizing activity.