Infection with the Hepatitis C virus (HCV) represents a major world-wide health problem. HCV is a liver-tropic RNA virus, and RNAi based treatments have been proposed as a new therapeutic modality. We are developing short-hairpin RNAs to elicit an RNAi response against the HCV genome. Robust expression of shRNAs from ubiquitous Pol III promoters has been demonstrated by various groups and authors. However, the potential for off-target effects makes it desirable to limit expression of shRNAs to the tissue of interest. Toward this goal we have evaluated liver-specific Pol II promoters for the expression of shRNAs. Most, if not all, transcripts generated by RNA pol II polymerase are capped. The 3' ends of these transcripts are usually polyadenylated. The presence of the Cap and/or the polyA tail may negatively affect the activity of Dicer leading to inactive, or low level active shRNAs. Several HCV specific shRNAs were cloned at variable distances downstream of a liver-specific promoter, such that transcription initiation occurred between 1 and 8 nucleotides from the start of the hairpin sequence. Termination of transcription was accomplished using either synthetic poly A sequences (based on the rabbit beta globin terminator, SPA), the U1 box terminator, or the minimal polyA cassette (as described by Xia et al (2002) Nat Biotech 20: 1006-1010). These constructs were evaluated in Huh7 cells by co-transfection with an HCV-luciferase fusion reporter. Transcription of the shRNA from the +2, +3, or +4 position resulted in 60-70 % inhibition of luciferase activity, similar to what has been observed using Pol III promoters. The level of silencing was similar regardless of the terminator used. These analyses have defined the optimal configuration for liver specific expression of anti-HCV short hairpin RNA transcripts. Use of these constructs should limit potential off target effects, without sacrificing high level hepatic expression. In vivo evaluation of these constructs is underway, with the ultimate goal of delivering these potential liver-specific HCV therapeutics to the liver with either viral or non-viral delivery vectors.