The genome of flaviviruses consists of an infectious single-stranded RNA molecule which contains a type 1 cap structure at the 5'-terminus. The cap is synthesized by RNA triphosphatase, guanylyltransferase and methyltransferase. Since flaviviruses replicate in the cytoplasm, it can be assumed that these functions are performed by virus-coded proteins. We previously showed that subtilisin treatment of membranes isolated from cells infected with the West Nile flavivirus results in release of a 50 kDa molecular weight fragment of the viral nonstructural protein NS 3. This so-called p50-S protein contains the residue gly (168) of NS 3 at the amino-terminus and represents an RNA-stimulated NTPase. In the present report we present experimental evidence which indicates that the p50-S protein also contains the active site of an RNA triphosphatase. The activity specifically cleaves the beta,gamma-triphosphate bond at the 5'-terminus of RNA. The localization of NS 3 protein sequence elements with known functions indicates that this multifunctional protein contains a protease in the amino-terminal part, a helicase in the central region and the RNA triphosphatase in the carboxy-terminal domain. An amino acid sequence element which may be involved in recognition of the 5'-terminal RNA triphosphate is tentatively identified. A homologous element may be present in the vaccinia virus-coded RNA triphosphatase.