A method for quantifying hepatitis C virus (HCV) RNA in serum using reverse transcription-polymerase chain reaction (RT-PCR) followed by slot-blot hybridization with a specific, digoxigenin-labeled probe was developed. Using RNA synthesized from cloned HCV cDNA as a standard, serum concentration of HCV RNA above 10 copies/ml can be quantitatively determined. To compare this method with branched DNA (bDNA) assay, 45 serum samples from 26 patients with newly acquired acute hepatitis C (n = 16) or hepatitis C with acute exacerbation (n = 10) were submitted to both assays. HCV RNA in 30 (67%), 12 (27%) and three (6.7%) samples can be quantitatively determined by both, either and none of the two assays, respectively. Using a standardized qualitative HCV RNA detection test (Amplicor HCV test) as a reference, 1 and 0 false positive results were found by bDNA and this assay, respectively. This quantitative assay using RT-PCR and a digoxigenin detection system was comparable to bDNA assay. Since a false positive result was rarely found, this technique can be used as a first line test to screen a large number of samples rapidly and economically.