Previous reports suggest that the hepatitis C virus (HCV) genome RNA terminates with homopolymer tracts of either poly(U) or poly(A). By ligation of synthetic oligonucleotides followed by reverse transcription-PCR, cDNA cloning, and sequence analysis, we determined the 3'-terminal sequence of HCV genome RNA. Our results show that the HCV 3' nontranslated region consists of four elements (positive sense, 5' to 3'): (i) a short sequence with significant variability among genotypes, (ii) a homopolymeric poly(U) tract, (iii) a polypyrimidine stretch consisting of mainly U with interspersed C residues, (iv) a novel sequence of 98 bases. This latter nucleotide sequence is not present in human genomic DNA and is highly conserved among HCV genotypes. The 3'-terminal 46 bases are predicted to form a stable stem-loop structure. Using a quantitative-competitive reverse transcription-PCR assay, we show that a substantial fraction of HCV genome RNAs from a high- specific-infectivity inoculum contain this 3'-terminal sequence element. These results indicate that the HCV genome RNA terminates with a highly conserved RNA element which is likely to be required for authentic HCV replication and recovery of infectious RNA from cDNA.