Abstract The hairpin ribozyme is a catalytic RNA capable of specifically cleaving a variety of substrate RNA sequences in either a cis or a trans reaction. The trans reaction has favorable catalytic and physical properties that make this ribozyme a potentially powerful in vivo regulator of gene expression. The ribozyme itself can be engineered to specifically cleave long RNA substrates by recognizing and cleaving a 14- to 20-nt target sequence. For certain sequences the reaction is efficient under mild, near physiological, conditions; however, this efficiency varies with the substrate and must be determined for each particular target. Substrate is cleaved at the * in the N*GUC sequence. Substrates containing a SN*GUC sequence, where the S is G or C, are cleaved most efficiently. To cleave heterologous RNA the hairpin ribozyme is engineered to base pair to the sequences flanking the N*GUC. The 5′ flanking sequence is fixed in length to 4 bp and the 3′ flanking sequence is variable, but the best catalytic efficiency is normally obtained when it is between 6 and 10 nt long. The ribozyme efficiently cleaves this substrate to generate the corresponding 5′ cleavage fragment and 3′ cleavage fragment. The 5′ cleavage fragment has a 2′,3′-cyclic phosphate 3′ terminus at the site of cleavage, and the 3′ cleavage fragment has a 5′-OH at the site of cleavage. The hairpin ribozyme has been engineered and shown to cleave targets in vivo . It has successfully been used to specifically down-regulate gene expression in mammalian cells acting as a catalytic antisense moiety.