Combination therapy with interferon-alpha (IFN-alpha) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20-30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1-407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 +/- 13 vs 69.4 +/- 22; P < 0.01). Subsequently, the effect of IFN-alpha plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection.