We developed a simple single tube procedure using the PCR (polymerase chain reaction) for the detection of hepatitis C virus RNA. The entire reaction from RNA extraction to PCR occurred in one tube; this was made possible by the use of more than 1,000 micrograms/ml of proteinase K for RNA extraction, instead of the acid-guanidinium thiocyanate-phenol-chloroform method. All necessary reagents were added to the tube, and PCR products were not removed from the tube until the end of PCR, although opening of the tube during the procedure could not be avoided. Therefore, cross-contamination which might theoretically take place during transfer of products between tubes never occurred. This method detected about 1 chimpanzee infectious dose of HCV in H strain plasma.