RNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy.