The 3'-terminal nucleotides of the flavivirus genomic RNA form conserved secondary structures that may function as cis-acting signals for RNA replication. Here we provide evidence for the existence of a conserved pseudoknot structure at the 3' terminus of the flavivirus genomic RNA. A truncated version of the West Nile virus (WNV) 3'-terminal RNA sequence was used as the model for these studies. Circular dichroism spectra indicated the presence of a highly structured RNA conformation with a significant amount of A-form helix. Ribonuclease probing not only confirmed the presence of the predicted secondary structure, which consists of a long stem-loop (SL1) and a shorter stem-loop (SL2), but also suggested that base pairing occurs between nucleotides in the loop of SL2 and those in an internal loop strand located on the 5' side of SL1. Analysis of three mutant RNAs further supported the existence of pseudoknot interactions. UV-melting analysis of the WNV 3' model RNA showed three transitions with significant hyperchromicity at approximately 46, 62, and 79 degrees C. UV-melting analysis with either SL1 or SL2 RNA alone suggested that the 62 and 79 degree C transitions represent the unfolding of SL2 and SL1, respectively. The 46 degree C transition is most likely due to the opening of the proposed tertiary structure. A similar melting curve was obtained for another flavivirus (dengue-3 virus) 3'-terminal RNA, providing further support for the conservation of the structure among flaviviruses. Molecular modeling of the RNA indicated that a pseudoknot structure is a stereochemically and energetically reasonable model for the 3' terminus of flavivirus genomic RNA.