A competitive reverse transcription PCR (cRT-PCR)-based assay for the quantitative detection of hepatitis C virus (HCV) viremia was developed, optimized, and applied to the direct molecular analysis of clinical samples from nine patients with persistent HCV infection. As for other competitive PCR-based applications, this method consists of the reverse transcription and subsequent amplification of two RNA species in the same tube: the wild-type template (to be quantified) and a known amount of a modified synthetic template. These templates have identical primer recognition sites and very similar (but not identical) sizes, thus allowing direct detection of both template species after gel electrophoresis and ethidium bromide staining. The results obtained by this cRT-PCR application for testing clinical samples from HCV-infected patients mainly indicate that the competitive approach reaches the degree of sensitivity (fewer than 5 HCV RNA molecules per 100 microliters) necessary to evaluate viral load in all HCV-infected patients, independently of clinical conditions, and that this technique is flexible enough to quantify highly divergent levels of cell-free HCV genome copy numbers in biological samples. Interestingly, we observed a sample-to-sample variation in the loss of detectable HCV genome molecules in serum in comparison with that in plasma from the same patient, thus indicating that serum specimens, although widely used in the past few years for qualitative molecular investigation of HCV-infected patients, cannot be used to obtain reliable quantitative data on HCV viremia from these patients.