Endonucleolytic hammerhead ribozymes have advantages of inhibiting gene expression by acting specifically, independently of cellular pathways, and within all cell compartments. However, there are concerns about inefficient silencing because of reduced intracellular cleavage of target RNA by ribozymes. To enable production of defined single-unit ribozymes and thereby increase effectiveness, we developed self-cleaving multimeric cassettes that generate several trans-acting ribozyme units from a single transcript. cis and trans ribozyme cleavage, as assessed in vitro against three different sites within the X sequence of hepatitis B virus (HBV), occurred efficiently and precisely according to predictions deduced from the ribozyme designs. Significant knockdown of markers of viral replication in transfected cultured liver-derived cells was achieved by multiribozyme Pol II expression cassettes. To assess silencing efficacy of RNA prepared in vitro, transcription and cis cleavage reactions were carried out to prepare defined single-unit ribozymes. Transfection of ribozyme RNA was capable of inhibiting HBV surface antigen secretion from liver-derived cells without associated elevation of interferon-alpha or interferon-beta secretion into the culture upernatants. The approach described here is potentially useful for several applications, such as generation of RNA interference (RNAi) effectors, which require rapid and inexpensive generation of defined RNA sequences.