Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30. 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9-21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9-21, 10-24, 151-165, 216-232, 282-301 and with peptide 340-354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10-24, 151-165, 216-232, 244-267, 260-274, 270-284, 260-284, 282-301, 300-314, 340-354, 348-362 and 355-369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10-24, which covers the same epitope as peptide 9-21, neutralized HSV-1 in vitro.