The terminal noncoding regions of viral RNA genomes are presumed to contain signal sequences and sometimes also secondary structures involved in regulating viral RNA synthesis. Such signals would be expected to be highly conserved among related viruses. In order to identify replication signal features for flaviviruses we have compared the 3'-terminal nucleotide sequences of West Nile virus (WNV), Saint Louis encephalitis (SLE) virus, and yellow fever virus (YFV) genome RNAs. The existence of a stable 3'-terminal secondary structure was previously predicted by a cDNA sequence obtained from YFV genome RNA. We have confirmed the existence of this structure by direct RNA sequencing methods. Even though the size and shape of the 3'-terminal secondary structure is highly conserved, sequence conservation is restricted to the loop regions of the secondary structure and to 27 nucleotides immediately adjacent to the 5' side of the structure. The regions of conserved sequence represent likely signals for viral polymerase recognition and binding. However, the preservation of the configuration of the secondary structure by a means other than sequence conservation indicate that this structure is important for the survival of the virus. A WNV mutant, which replicates progeny genome RNA more efficiently than parental WNV, was found to have a 3'-genomic sequence identical to that of its parent virus. The sequence change conferring the phenotype of this mutant is therefore located in another region of the genome.