The 5' untranslated region of hepatitis C virus RNA forms an extensive secondary structure including several hairpin motifs and mediates translation initiation by an internal ribosome entry site-dependent pathway. We report, here, an extensive mutagenesis analysis of a highly conserved tetraloop in the 5' untranslated region of hepatitis C virus, namely hairpin IIIe (295'-GAUA-298'). Our results demonstrate that hairpin IIIe is essential for the internal ribosome entry site function. Moreover, they indicate the importance of the primary structure of this motif because mutations in all four nucleotides of the loop caused a severe loss of internal ribosome entry site activity. These data represent the first experimental evidence for the functional significance of tetraloops in internal ribosome entry site-driven translation of hepatitis C virus.