A combination of phage peptide display library mapping and pepscanning, with both murine monoclonal antibodies and a panel of well-characterized human sera, have been used in order to define type-specific epitopes of glycoprotein G of herpes simplex virus type 2 (HSV-2) (gG2). Both techniques revealed an immunodominant region of gG2, centred around amino acids 525-587 of the uncleaved gG2 molecule. A soluble peptide, equivalent to amino acids 551-570, when used as antigen in an ELISA format was recognized by three out of five murine MAbs and by 20/26 (77%) Western blot anti-HSV-2-positive human sera, but by only 1/63 Western blot anti-HSV-2-negative sera (specificity, 98%). The sensitivity of detection of human anti-HSV-2 antibodies was increased to 90% using a peptide derived from this region, presented on a nitrocellulose membrane. This highly antigenic and type-specific domain of gG2 is located at the junction between the 'unique' region of gG2 and its C-terminal end, which has approximately 50% identity with gG1. A second antigenic region of gG2, amino acids 351-427, which lies within the 'unique' part of gG2, was also identified by both techniques employed in this study and is recognized by a proportion of anti-HSV-2-positive sera. These findings demonstrate the feasibility of developing a peptide-based type-specific assay for the detection of anti-HSV-2 antibody in human sera based on type-specific epitopes of gG2 and have implications for the understanding of the three-dimensional topography of gG2.