The catalytic domain of a hammerhead ribozyme was Incorporated Into a 413 nucleotides long antisense RNA directed against the 5′-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to + 634). The resulting catalytic antisense RNA was shown to cleave Its target RNA In vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA In vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytlcally active domain resulted In 4 to 7 fold stronger Inhibition of HIV-1 replication as compared to the parental antisense RNA and the Inactive mutant. Kinetic and structural studies performed In vitro indicated that the ability for double strand formation was not changed In catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA Is a crucial prerequisite for the observed Increase of inhibition of the replication of HIV-1.