Hammerhead ribozymes are potentially important tools for suppressing intracellular expression of unwanted RNAs. However, the reports that exist on their activity against different targets have described mixed success. As an initial step towards developing a rapid and effective system for in vivo testing of ribozymes, two human immunodeficiency virus type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, RzPro directed against the protease (Pro) coding region and RzRT directed against the reverse transcriptase (RT) coding region, were designed and tested in Escherichia coli. Both ribozymes displayed similar efficiencies in cleaving their target RNAs in vitro. RNA polymerase chain reaction was adapted to demonstrate the in vivo cleavage of RzPro and RzRT target sites. The resultant drop in HIV-1 RT activity was measured as well. The degree of suppression of RT activity was more apparent in vivo in cells expressing RzRT. The RT activity in cells expressing RzRT was shown to decrease by up to 96%. This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti-HIV-1 gene therapy, (ii) ribozymes with improved stability and catalytic activity, and (iii) cofactors, if any that could enhance ribozyme activity in vivo.