A rapid and reproducible enzyme-linked immunosorbent assay (ELISA) is described for determining antibodies in human sera against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The sera were absorbed for 30 min with heterologous virus-infected-cell extracts to remove cross-reacting antibodies and then were applied to ELISA plates containing the target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD and HSV-2 glycoproteins gD and gF. The absorbance index, defined as the ratio of A414 generated by a serum sample absorbed with a heterologous virus-infected-cell extract versus the A414 of a serum sample absorbed with an uninfected-cell extract, was used to determine the presence or absence of antibodies to HSV-1 and HSV-2. Results of the ELISA for detecting antibodies against HSV-2, when compared with results obtained for the same sera by the microneutralization test, showed an index of overall agreement of 91%. Results of the ELISA for detecting antibodies against HSV-1, when compared with microneutralization test results for sera negative for HSV-2 antibodies but positive for HSV antibodies by ELISA, showed an index of agreement of 99%.