Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5' end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5'-RNA triphosphatase activity, suggesting its role in the synthesis of the 5'-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5'-RNA triphosphatase activity that cleaves the gamma-phosphate moiety of the 5'-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside triphosphatase and 5'-RNA triphosphatase activities of NS3 share a common active site.