Activating RNA interference to achieve specific gene silencing has shown promise for the development of RNA-based treatment of chronic hepatitis B virus (HBV) infection. To further this approach, we assessed the efficacy of expressed long hairpin RNAs (lhRNAs) that target the conserved HBx open reading frame of HBV. As substrates for Dicer, lhRNAs have the potential to generate multiple short interfering RNAs (siRNAs) to enable simultaneous targeting of different sites. Two U6 Pol III vectors were constructed that encode anti-HBV lhRNAs with a 62 base pair stem sequence containing multiple G:U pairings. Assessment in transfected cultured cells and also in vivo using the murine hydrodynamic injection model showed that one of the lhRNA vectors (lhRNA 1) diminished markers of virus replication by 70-90% without evidence of interferon response induction. Greatest silencing efficacy was observed for targets that are complementary to sequences located at the base of the hairpin stem and this correlated with a higher concentration of siRNAs derived from this region of the lhRNA. Although lhRNA 1 has the advantage of targeting a greater viral sequence, incomplete cellular processing may result in unequal silencing across the span of the viral target RNA.