Based on recent LightCycler techniques developed for the quantitation of serum HCV RNA, we have developed a quantitative method for the intracellular hepatitis C virus (HCV) RNA using LightCycler PCR. A simple real-time PCR assay, based on the SYBR Green I dye and LightCycler fluorimeter and with no probe requirement, is described. In the presence of 0.5 microg of cellular RNA, it was demonstrated that as few as 25 copies of HCV RNA could be specifically detected with a set of primers that amplify a 144-base pair sequence unique to the 5'-noncoding region of HCV RNA. We demonstrated that this method was useful for the evaluation of antiviral reagents using HCV-infected human cultured cells.