We compared two primer sets (A: 167bp, B: 269bp) derived from highly conserved domains within the 5' noncoding region (5'NC) of the hepatitis C virus (HCV) genome for their ability to detect HCV-RNA in a nested cDNA polymerase chain reaction assay (nested-PCR) in sera from 31 patients suspected of having HCV infection. Seventeen (54%) of 31 patients were positive for HCV-RNA with both primer sets. Using primer set A, 14(93%) of 15 samples with positive and 3(19%) of 16 samples with negative anti-HCV antibody test gave positive results for HCV-RNA. With primer set B, 15(100%) of 15 antibody positive samples and 2(13%) of 16 negative samples were positive for HCV-RNA. One antibody negative sample from a patient with alcoholic liver cirrhosis was positive for HCV-RNA only with primer set A. Another sample with positive antibody test, from a patient with chronic renal failure, was positive for HCV-RNA only with primer set B. A combination of more than one set of primers directed to the highly conserved 5'NC region, as well as proper selection of the exact nucleotide sequences, are important in improving the detection rate of HCV-RNA by PCR in serum of infected patients.