We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severely defective as templates for either polymerase. We also evaluated the infectivity of substitution mutant WNV genome-length RNAs. All WNV RNAs containing the DV2 3'SL were unable to replicate. However, WNV RNAs containing substitutions of the 5'NCR, the capsid gene, and/or 3'NCR nt sequences upstream from the WNV 3'SL, by the analogous DV2 nt sequences, were infectious. Combined results suggested that replication was not dependent upon species homology between the 3'SL and NS5.