The establishment of tissue culture systems that consistently support high levels of HCV replication is a priority not only for elucidating the replication cycle of the virus but also for screening of putative antiviral compounds. Although many groups have reported virus production in various tissue culture systems (Table 11.1), the low levels of virus produced over time has necessitated using RT/PCR for detection (Ch. 12). Given that HCV is closely related to flaviviruses (Miller & Purcell, 1990) (Table 2.1), it has been assumed that the replication cycle of HCV, like that of flaviviruses, would contain a minus strand intermediate (Chambers et al., 1990; Monath, 1990). The finding of HCV minus strand RNA in tissue culture cells (Table 11.1) and in liver samples (Fong et al., 1991b; Gerber et al., 1992; Negro et al., 1998) from infected patients is consistent with a role for minus strand in the replication cycle, although this has yet to be formally proven. From a technical perspective, detection of minus strand RNA is problematic at best, since it involves using strand-specific primers, which may also randomly prime on the viral plus strand (or on cellular mRNAs), resulting in false-positive results. In tissue culture systems that depend upon infection of susceptible cells with virus from an infectious serum, it is not always clear whether the RT/PCR results reflect de novo production of RNA and/or detection of RNA extracted from virus particles that simply adhered to the tissue culture cells.