UV cross-linking analyses were performed in an attempt to determine cellular protein-viral RNA interactions with the 3' untranslated region (3' UTR) of the hepatitis C virus RNA genome. Two cellular proteins, with estimated molecular masses of 58 kDa (p58) and 35 kDa (p35), respectively, were found to specifically bind to the 3' UTR. The p58 protein was determined to be the polypyrimidine tract-binding protein. In addition to binding to the conserved 98 nucleotides (nt) of the 3' UTR, p58 also binds to the poly(U) tract of the 3' UTR. The p35 protein was found to interact only with the poly(U) tract of the 3' UTR. These conclusions are supported by the following findings: (1) p58, and not p35, binds to the 3' end conserved 98 nt, (2) both p58 and p35 bind to a 3' UTR RNA with a deletion of the conserved 98 nt, (3) the 98-nt deletion mutant 3' UTR competed out both p58 and p35 binding, (4) a poly(U) homopolymer competed out both p58 and p35 binding, (5) a 3' UTR RNA with deletion of the poly(U) tract competed out only p58 binding but not p35 binding, and (6) an RNA containing the variable region of the 3' UTR with a deletion of both poly(U) tract and 98 nt failed to compete for binding of either p58 or p35. Interaction of these cellular proteins with the HCV 3' UTR is probably involved in regulation of translation and/or replication of the HCV RNA genome.