BACKGROUND A completely modified phosphorothioate antisense oligodeoxynucleotide (cS-ODN 4) directed against nucleotides 326-348 of the hepatitis C virus (HCV) 5' non-coding region (NCR) efficiently inhibits viral gene expression. As cS-ODN exerts undesired side-effects in vivo, we synthesized partially modified ODN 4 that contained only six modified nucleotides which are located at the ODN termini or are scattered along the molecule. The tested modifications were polar phosphorothioates (S) and non-polar methyl- (M) or benzylphosphonates (B). RESULTS In an in vitro translation system, specific inhibition of HCV gene expression by M-ODN 4 or B-ODN 4 was observed if terminally modified ODN were used; the maximal inhibition was 92.3% +/- 1.9% and 87.1% +/- 3.7%, respectively, at 10 microgram mol L-1 concentration. S-ODN 4 specifically suppressed viral translation irrespective of the location of the modifications, resulting in a maximal inhibition of 86.3% +/- 3.3%. For all terminally modified ODNs the therapeutic index was high, with tB-ODN 4 the second best at 3.8. Inhibition correlated with efficient RNase H-associated cleavage of target RNA. In transient co-transfection experiments of HepG2 cells with a reporter gene construct and the ODN, terminally modified B-ODN 4 was the most effective and specific inhibitor. At a concentration of 5 microgram mol L-1 the suppression of HCV translation was 96.3% +/- 0.7%. CONCLUSIONS These data demonstrate that terminally modified B-ODN 4 is a potent inhibitor of HCV gene expression in vitro and in HepG2 cell culture and may be valuable for future antiviral treatment.