The genetic diversity of dengue (DEN) virus was explored using two South American DEN-1 virus strains isolated from viremic human sera. DEN-1 virus strains BR/90 and FGA/89 were selected on the basis of their membrane fusion properties in mosquito cell cultures. Infection of mosquito cell lines with BR/90 virus strain induced a cytopathic effect characterized by syncytium formation whereas no cytopathic changes were observed with FGA/89. Cell-to-cell fusion experiments indicated that the fusogenic activity of FGA/89 required a lower pH than BR/90. Immunoreactivity analysis of the DEN-1 envelope (E) protein with monoclonal antibodies revealed a minor difference between the antigenic structures of FGA/89 and BR/90 virions. FGA/89 was less neurovirulent than BR/90 for newborn mouse. To determine the genetic origin of these modifications, the amino acid sequences of the structural proteins from these virus strains were compared. One amino acid difference was found within the carboxy-terminal domain of protein C. Five amino acid substitutions were found in the E proteins at positions 96, 180, 297, 379, and 473. Changes at positions 96, 297, and 379 map within two overlapping antigenic domains of protein E. These limited amino acid differences in the E protein could affect the biological properties and the antigenicity of the DEN virion.