The 5'-noncoding region of hepatitis C virus (HCV) genomes is highly conserved. A two-stage polymerase chain reaction (PCR), involving two pairs of primers deduced from the 5'-noncoding region of the HCV genome, was developed for a sensitive and specific detection of HCV RNA. The first stage of PCR was performed for 35 cycles with primers capable of multiplying fragments of 221 base pairs. PCR products in samples negative for HCV RNA were subjected to the second stage of PCR for 30 cycles with primers located internal to those employed in the first stage of PCR. The two-stage PCR detected up to 10 chimpanzee infectious doses/ml of HCV, and HCV RNA in 11 (92%) of 12 sera from patients with chronic non-A, non-B hepatitis without detectable antibodies to HCV by a commercial assay kit. Primers from the 5'-noncoding region of the HCV genome would be suitable for detecting HCV RNA by PCR, since the other regions of the HCV genome diverge extensively in sequence because of its nature as an RNA virus.