Human antibody responses to specific polypeptides of herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) were assessed in serial serum specimens from 18 infected patients by immunoblot technology. Nine patients had HSV-1 infections (six genital and three oral) and nine had HSV-2 genital infections. Antibodies to homologous and heterologous HSV antigens were studied and correlated with total microneutralization and enzyme-linked immunosorbent assay antibodies as well as correlated directly to purified glycoproteins. The data indicated a sequential appearance of antibodies to specific polypeptides, according to virus type and site of infection. After HSV-1 infection, the initial response was to glycoprotein B, but the same was not true for HSV-2 infection, where the initial response appeared to be to the type-specific glycoprotein G. A difference in sequential appearance of antibodies for the two viruses indicated greater reactivity to lower-molecular-weight polypeptides after genital infection, irrespective of type, in contrast to nongenital HSV-1 infections. The antibody responses for selected sera to purified glycoproteins B and D were verified by enzyme-linked immunosorbent assay antibody determinations.