RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (-)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (-)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (-)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (-)-strand RNA synthesis but neither the (-)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (-)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (-)-strand RNA synthesis but not for (+)-strand RNA synthesis.