Translation initiation of hepatitis C virus (HCV) RNA genome is mediated by an internal ribosome entry site (IRES). To further comprehend the mechanism of translation initiation of HCV RNA, we investigated the importance of two unstructured, highly conserved, single-stranded pyrimidine-rich sequences located immediately upstream of domain II (nt38-43) and between domains II and III (nt120-125) in HCV translation. A series of defined mutations was engineered and introduced into a dicistronic vector in order to assess their impact on in vitro translation. Our data indicated that nucleotide sequence 38-43 is not essential for HCV translation. In contrast, mutational analysis of the second sequence motif (nt120-125) suggested that this region was important for maintaining the proper structure within the IRES element although the primary sequence itself was not critical for IRES function. More importantly, it appeared that mutations which allowed juxtaposition of neighboring bases (nt112-119) to the pseudoknot structure, were detrimental to translation initiation.