We used computer analysis to study hydrophilicity, homology, surface accessibility, molecular flexibility, and secondary structure of the deduced amino acid sequence of the flavivirus envelope (E)-glycoprotein. Using the results, we modified the E-glycoprotein antigenic structure proposed by Nowak and Wengler (1987, Virology, 156, 127-137). Our model predicts considerable overlaps in the previously defined domains. We have prepared 11 synthetic peptides from the deduced amino acid sequence of the E-glycoprotein of Murray Valley encephalitis (MVE) virus and analyzed their immunogenicity. Peptides derived from the redefined R1 and R2 domains elicit antiviral antibody. Nine of these peptides are recognized by polyclonal antiviral antibodies; however, none are consistently recognized by monoclonal antibodies. Peptides derived from the R1 domain demonstrate MVE virus specificity, and 1 peptide elicited low-level virus neutralizing antibody. Spatial overlap of the domains was defined by competitive binding assays between antipeptide antisera and radioactive monoclonal antibodies. These results indicate that synthetic peptides aid in defining flavivirus antigenic structure, and may serve as possible type-specific diagnostic reagents.