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The nucleic acid sequence of the putative 5'-untranslated (5PUT) region of hepatitis C virus (HCV), determined for samples obtained from a variety of geographic origins, was found to be over 98% conserved among all isolates. On the basis of this signature sequence for HCV, a viral RNA assay was developed by using cDNA synthesis with reverse transcriptase, followed by polymerase chain reaction (PCR). The new assay was compared with the Ortho-Chiron C100-3 HCV enzyme-linked immunosorbent assay to research radioimmunoassays for antibodies to the C33c and C22 HCV antigens and to the first reported set of HCV PCR primers designed from the NS3 domain. Plasma samples from 16 Japanese patients with non-A, non-B hepatitis (NANBH) and 16 immunoassay-positive blood donors from the United States were investigated. The 5PUT PCR primers were found to be superior to the NS3 primers in sensitivity and specificity (15 of 25 versus 3 of 25 of the C100 enzyme-linked immunosorbent assay-positive samples, respectively). Samples from two C100-negative patients with acute NANBH were found to react with the 5PUT primers but not with the NS3 primers. Also, two of three patients with chronic NANBH converted from reverse transcriptase PCR positive to negative after interferon treatment. Although the clinical significance of the presence or absence of HCV RNA in samples from patients is not fully understood, the use of probes and primers from the 5PUT region (as opposed to primers from other segments) should not lead to false-negative results due to nucleic acid sequence variations in viral isolates.