A rapid and simple method using the polymerase chain reaction (PCR) was devised for the co-amplification and simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1), specific sequences in the same serum sample. Genomic RNA was extracted from 13 blood donor sera that were reactive in ELISA for both anti-HCV and anti- HIV-1. The extracted RNA was reverse transcribed into cDNA and amplified using nested primer pairs (SN01, SN02, SN03 and SN04) based on the HCV prototype sequence of clones 37b and 81 for HCV, and SK 38/39 for HIV-1 simultaneously. PCR products were analyzed by liquid hybridization and Southern blot hybridization with 32P end-labeled oligonucleotide probes from the regions between the two primer pairs, excluding the primer sequences. The primer pairs were able to detect as few as 1-3 copies and 10-20 copies of genomic RNA for HCV and HIV-1, respectively, when compared with 8E5 cells as the single copy standard. HCV-RNA was detected in all 13 (100%) samples tested; HIV-RNA was detected in 11 (85%) samples. The ability to coamplify specific sequences from two different viral genomes in the same reaction mixture could facilitate the simultaneous detection of multiple viral sequences, thus offering the possibility of simultaneous diagnosis of more than one viral agent in serum samples of symptomatic and asymptomatic individuals.