We compared the relative sensitivities of first-and-second generation branched nucleotide assays (Quantiplex HCV RNA 1.0 and 2.0, respectively, Chiron, Emeryville, Calif) for the detection of hepatitis C virus (HCV) RNA to that of a commercially available quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method (Monitor, Roche Molecular Systems, Nutley, NJ) in 53 patients with chronic hepatitis C. The sensitivities of the second-generation branched DNA (bDNA) and RT-PCR assays were similar (91% and 92%, respectively), and both were significantly more sensitive (P < .001) than the first-generation method. Moreover, both assays detected HCV RNA in all 11 patients with type 2a, 2b, or 3a genotypes vs 45% with the HCV RNA 1.0 bDNA assay. We examined 174 serum samples by the bDNA 2.0 and RT-PCR assays. Major quantification differences were noted on a given specimen with the RT-PCR method reporting values an average 41-fold lower (range, 0-703-fold) than those obtained with the bDNA assay. We conclude that both methods can be used to detect HCV RNA in patients who are infected with the genotypes that are most commonly encountered in the United States. The HCV RNA 2.0 bDNA assay may offer advantages when attempting to quantify high-level viremia.