The 5′ untranslated region (5′UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5′UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5′UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5′UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem–loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem–loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings,in vitrotranslation inhibition assays using RNA fragments of the 5′UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these twocis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5′UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. Thesecis-acting translational elements may serve as potential targets for the antiviral action of oligomers.