Hepatitis C virus (HCV) translation is mediated by an internal ribosome entry site (IRES) located at the 5' end of the genomic RNA. The 3' untranslatable region (3'UTR) stimulates translation by the recruitment of protein factors that simultaneously bind to the 5' end of the viral genome. This leads to the formation of a macromolecular complex with a closed loop conformation, similar to that described for the cap-translated mRNAs. We previously demonstrated the existence of a long-range RNA-RNA interaction involving subdomain IIId of the IRES region and the stem-loop 5BSL3.2 of the CRE element at the 3' end of the viral genome. The present study provides evidence that the enhancement of HCV IRES-dependent translation mediated by the 3'UTR is negatively controlled by the CRE region in the human hepatoma cell lines Huh-7 and Hep-G2 in a time-dependent manner. Domain 5BSL3.2 is the major partner in this process. Mutations in this motif lead to an increase in IRES activity by up to eightfold. These data support the existence of a functional high order structure in the HCV genome that involves two evolutionarily conserved RNA elements, domain IIId in the IRES and stem-loop 5BSL3.2 in the CRE region. This interaction could have a role in the circularisation of the viral genome.