To further explore the controversial potential for extrahepatic replication of hepatitis C virus (HCV), the highly strand-specific rTth method of reverse transcriptase PCR was used to examine sera, liver, peripheral blood mononuclear cells, and other extrahepatic tissues from HCV-infected chimpanzees and humans. Positive-strand HCV RNA was present in the liver at approximately 10-fold-higher levels than negative-strand HCV RNA. No negative-strand RNA was detected in peripheral blood mononuclear cells or other extrahepatic tissues despite the presence of abundant positive-strand RNA. These data demonstrate that within the limits of sensitivity of this highly strand-specific reverse transcriptase PCR method, no extrahepatic replication of HCV was detected.