Chronic hepatitis B virus (HBV) infection is endemic to several populous areas of the world and is frequently complicated by hepatocellullar carcinoma. Ribozymes can be designed to cleave target RNA sequences specifically and show promise for the treatment of HBV infection. Demonstration of intracellular inhibition of REV gene expression, essential to developing therapeutic ribozymes, has been the aim of this investigation. We generated two vectors encoding hammerhead ribozymes that target the HBx region of HBV. Plasmids containing intact HBV sequences or a modification in which the preS2/S region was replaced by DNA encoding enhanced green fluorescent protein (EGFP) were used to test ribozyme action in transfected cells. Both ribozymes inhibited surface antigen secretion and EGFP expression similarly. The measurement of EGFP expression is convenient to assess ribozyme action in situ and effective targeting of HBV sequences that are common to all HBV transcripts is potentially useful to develop strategies to counter HBV infection.