The lymphocytic choriomeningitis virus (LCMV) structural glycoproteins GP-1 (Mr 44K) and GP-2 (Mr 35K) are encoded on a single intracellular proteolytic cleavage precursor glycoprotein, GP-C (Mr 76K). We have used a series of synthetic peptides derived from the deduced amino acid sequence of LCMV GP-C to define an antigenic site containing two topographically overlapping epitopes. Three mouse monoclonal antibodies directed against two epitopes on GP-2 were assayed for binding in solution phase blocking and solid-phase enzyme-linked immunoadsorbant assays to a series of peptides representing the sequence of the intracellular precursor glycopeptide GP-C. Both epitopes were initially localized to a single peptide GP-C 370-382 (Cys-Asn-Tyr-Ser-Lys-Phe-Trp-Tyr-Leu-Glu-His-Ala-Lys) in the GP-2 segment of GP-C. Further analysis demonstrated that both epitopes were contained within a nine amino acid segment, GP-C 370-378, which contains five residues conserved among LCMV, Lassa, Pichinde, and Tacaribe viruses. Assays with N-terminal deletions from this sequence suggested that the minimal epitope recognized by the broadly cross-reactive monoclonal 33.6 (epitope GP-2a) consisted of five amino acids, GP-C 374-378 (Lys-Phe-Trp-Tyr-Leu). Reactivity of a second monoclonal, 9-7.9 (epitope GP-2B) but not 33.6, was abolished when substitution of tyrosine for phenylalanine was made at position 375 in the antigenic sequence corresponding to a naturally occurring sequence difference between LCM and Lassa viruses. Polyclonal sera from human cases and from animals experimentally infected with Junin, LCM, and Lassa viruses, respectively, bound to the antigenic peptide GP-C 370-382 but not to control peptides. As was the case with the monoclonals, this binding activity was abrogated by blocking with the antigenic peptide but not with control peptides in solution.