Previous studies have demonstrated reduced replication of the cell culture-adapted Dengue-2 virus (DENV-2) vaccine candidate, primary dog kidney (PDK)-53, compared with the parental DENV-2 strain, 16681, in C6/36 cells. Various DENV-2 mutants incorporating PDK-53 substitutions singly and in combination into the 16681 genetic backbone were used to identify the genetic basis for impaired replication of the vaccine candidate in vitro in Aedes aegypti cell culture (Aag2 cells) as well as the reduced in vivo infectivity and transmissibility within Ae. aegypti infected by intrathoracic inoculation. 5' untranslated region (UTR-c57t) and nonstructural protein 1 (NS1-G53D) mutations were required to completely attenuate in vitro replication. In contrast, incorporation of the PDK-53-specific NS3-250V mutation into the 16681 virus resulted in reduced replication in mosquitoes but had no effect on in vitro replication. Further, reversion of the PDK-53 NS3-250 site to that of the wild-type 16681 virus (NS3-V250E) failed to increase either in vitro or in vivo replication. Intrathoracic inoculation of Ae. aegypti with mutants containing the PDK-53 NS1 substitution exhibited in vivo replication indistinguishable from the parental PDK-53 virus, implicating this mutation as the dominant determinant for impaired mosquito replication of the PDK-53 candidate; however, further attenuation of in vivo replication was magnified in mutants including the additional 5'UTR-c57t mutation.