Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5′- and 3′-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5′- and 3′-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNAVal, U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3′/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5′- or 3′-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3′-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNAVal as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3′-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNAVal-driven Rzs and U6-derived siRNAs both directed against highly conserved 5′- and 3′-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.