The disease dengue (DEN) is caused by four genetically and serologically related viruses termed DENV-1, -2, -3, and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3. The ED3 contains the DENV type-specific and DENV complex-reactive (epitopes shared by DENV 1-4) antigenic sites. In this study the epitopes recognized by four DENV complex-reactive monoclonal antibodies (MAbs) with neutralizing activity were mapped on the DENV-3 ED3 using a combination of physical and biological techniques. Amino acid residues L306, K308, G381, I387, and W389 were critical for all four MAbs, with residues V305, E309, V310, K325, D382, A384, K386, and R391 being critical for various subsets of the MAbs. A previous study by our group (Gromowski, G.D., Barrett, N.D., Barrett, A.D., 2008. Characterization of dengue complex-specific neutralizing epitopes on the envelope protein domain III of dengue 2 virus. J. Virol 82, 8828-8837) characterized the same panel of MAbs with DENV-2. The location of the DENV complex-reactive antigenic site on the DENV-2 and DENV-3 ED3s is similar; however, the critical residues for binding are not identical. Overall, this indicates that the DENV complex-reactive antigenic site on ED3 may be similar in location, but the surprising result is that DENV 2 and 3 exhibit unique sets of residues defining the energetics of interaction to the same panel of MAbs. These results imply that the amino acid sequences of DENV define a unique interaction network among these residues in spite of the fact that all flavivirus ED3s to date assume the same structural fold.