In vitro translation experiments involving reticulocyte and HeLa cell lysates have demonstrated that translation initiation on hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal sequence (internal ribosomal entry site [IRES]) within the 5' noncoding region (5'NCR). Specific binding factors that bind to the HCV IRES were examined by UV cross-linking tests. A protein p57 (polypyrimidine tract binding protein [PTB]), known to be a binding factor that binds to picornavirus IRES, was found to bind to the HCV IRES. To investigate functions of this PTB in IRES-dependent translation initiation, hyperimmune antibodies to PTB (anti-PTB) were prepared by immunizing rabbits with purified recombinant PTB produced in E. coli. Anti-PTB IgGs were purified and used to deplete PTB in a cell-free protein synthesis system prepared from HeLa cells. In vitro translation experiments using PTB-depleted lysates suggested that host factor dependency of IRES is different in individual IRESs. As we showed previously, function of the IRES typical of group II HCV is more efficient than the IRES typical of group I HCV in a cell-free protein synthesis system from HeLa cells. Recombinant HCV IRESs were constructed and tested for their function in the in vitro system. Most recombinant IRESs showed an initiation activity of the more efficient typical group II IRES level or higher. This observation, together with the fact that group I HCV is the most common isolate of HCV worldwide, suggests that the efficiency of the IRES function of HCV may be generally suppressed in nature, and that the replication ability of HCV that permits its persistent infection in humans may require a delicate balance at a molecular level.