Qualitative detection of negative hepatitis C virus (HCV) RNA has been used widely to demonstrate HCV replication. However, relative quantitation of both positive and negative HCV RNA strands has never been reported for studying viral genome replication. A strand specific real-time PCR carried out in the highly conserved 5'-non-coding region of HCV genome and monitored either by the DNA binding dye SYBR Green I or by molecular beacons is described. Using these techniques, it was found that negative HCV RNA strand was a 100-1000 times less abundant than the positive strand in the liver of HCV infected patients.