Immunization of mice with a synthetic peptide that corresponds to a murine antibody-defined immunodominant domain of herpes simplex virus (HSV) glycoprotein D (gD) induced neutralizing antibodies against HSV types 1 and 2 and protected animals against a lethal challenge with HSV type 2 (Dietzschold, B., Eisenberg, R., Ponce de Leon, M., Golub, E., Hudecz, F., Varicchio, A. & Cohen, G. (1984) J. Virol. 52, 431-435). We report here that human peripheral blood T cells from HSV-seropositive and -seronegative adult donors are activated by this synthetic peptide in vitro. Interleukin-2-dependent T-cell lines established from these cultures respond specifically to peptides containing residues 1-23 of HSV gD and to a panel of overlapping peptides within this domain. The T-cell proliferative response was maximal when the majority of interleukin-2-propagated T cells were of the helper phenotype and the peptides were at least 16 amino acids long. Peptides of 8 or 12 amino acids from the carboxyl terminus were nonstimulatory. Peptide-activated T-cell lines from sero-negative donors less than 11 years old could be established in vitro, but most cells were of the suppressor/cytotoxic phenotype and demonstrated no antigen-specificity when tested with the panel of synthetic peptides.