Performance characteristics of a hepatitis C virus (HCV) RNA quantification assay comprised automated specimen extraction [COBAS AmpliPrep (CAP) using total nucleic acid isolation reagents (TNAI)], and real-time polymerase chain reaction [COBAS TaqMan 48 HCV with analyte-specific reagents (CTM48)] were determined. CAP TNAI/CTM48 performed linearly from approximately 2.0 to at least 6.7 log10 IU/ml for HCV genotypes (Gts) 1, 2, and 3. The limit of detection for the World Health Organization International Standard was 23 IU/ml. Variabilities ranged from 1.3 to 2.1%. Excellent quantitative agreement was observed in clinical samples using CTM48 and two different methods for HCV RNA extraction (CAP TNAI and BioRobot M48; regression line slope, 0.98; y-intercept, 0.11; R2, 0.98; mean difference, 0.003). Good agreement was also observed between CAP TNAI/CTM48 and COBAS Amplicor Monitor (regression line slope, 0.94; y-intercept, 0.08; R2, 0.96), although HCV RNA concentrations were on average greater by COBAS Amplicor Monitor (mean difference -0.27 log10 IU/ml). Better overall agreement was observed for Gt 1 than non-Gt 1 specimens when comparing extraction and quantification methods; however, no consistent genotype-dependent quantification bias was observed. These data suggest that CAP TNAI/CTM48 offers an alternative method for the quantification of HCV in plasma samples.