Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.