The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5′ nontranslated regions (5′NTRs) sharing short segments of conserved primary nucleotlde sequence. The functions of these 5′NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5′NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5′NTRs are highly conserved structurally, despite substantial differences in their primary nucleotlde sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5′NTR of HCV resulted in a model of the secondary structure of the HCV 5′NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3′ 200 bases of the 5′NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (Internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.