Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.