ABSTRACT Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of an infectious cell culture system using the genotype 2a isolate JFH-1. Taking advantage of this system in the present study, we investigated whether HCV infection leads to superinfection exclusion, a state in which HCV-infected cells are resistant to secondary HCV infection. To discriminate between viral genomes, we inserted genes encoding fluorescent proteins in frame into the 3′-terminal NS5A coding region. These genomes replicated to wild-type levels and supported the production of infectious virus particles. Upon simultaneous infection of Huh-7 cells, coreplication of both viral genomes in the same cell was detected. However, when infections were performed sequentially, secondary infection was severely impaired. This superinfection exclusion was neither due to a reduction of cell surface expression of CD81 and scavenger receptor BI, two molecules implicated in HCV entry, nor due to a functional block at the level of virus entry. Instead, superinfection exclusion was mediated primarily by interference at the level of HCV RNA translation and, presumably, also replication. In summary, our results describe the construction and characterization of viable monocistronic HCV reporter genomes allowing detection of viral replication in infected living cells. By using these genomes, we found that HCV induces superinfection exclusion, which is primarily due to interference at a postentry step.