We report on the synthesis and conformational properties of 2´-deoxy-2´,4´-difluorouridine (2´,4´-diF-rU) and cytidine (2´,4´-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially 'lock' the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2´,4´-diF-rU 5´-triphosphate (2´,4´-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2´,4´-diF-rUTP inhibited RNA synthesis in dinucleotide primed reactions, although with relatively high half maximal inhibitory concentrations (IC50 > 50 micromolar). 2´,4´-diF-U/C represent rare examples of 'locked' ribonucleoside mimics that lack a bicyclic ring structure.