T o determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRCdelta-luc, was constructed containing the 5'-noncoding region (5'-NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRCdelta-luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRCdelta-luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRCdelta-luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRCdelta-luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions.