We have compared the expression of the core gene of two different hepatitis C virus (HCV) isolates, HCV-1 and HCV-RH, using the in vitro translation assay. In the absence of the downstream E1 envelope protein sequence, a 16-kDa protein (P16) was the dominant protein product synthesized from the HCV-1 core gene sequence. On the other hand, a 21-kDa protein (P21) was the dominant protein product synthesized from the HCV-RH sequence. Domain-swapping and site-directed mutagenesis experiments indicated that codon 9 of the core protein coding sequence played a crucial role on the synthesis of the core protein: a lysine codon at this position led to the synthesis of P16 and an arginine codon at this position led to the synthesis of P21. For HCV-1 and HCV-RH, this codon encodes lysine and arginine, respectively. Further analyses indicated that P16 was likely co-amino-terminal with P21. In the presence of the downstream E1 envelope protein sequence and microsomal membranes, P16 as well as P21 were synthesized from the HCV-1 core gene sequence whereas P21 remained the only detectable protein product synthesized from the HCV-RH core gene sequence. These results indicate that the pathway leading to the synthesis of the HCV core protein may be more complicated than originally envisioned.