Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize pre-tRNA-like complexes between a 5' half tRNA(Arg) and a 3' half tRNA(Arg) with a 3' trailer, and can cleave the 3' half tRNA(Arg) after the discriminator nucleotide (Nashimoto M., 1996, RNA, 2:523-534). 3' tRNase cleaved the 3' half tRNA(Arg) in the presence of a 7-nt 5' tRNA(Arg) composed only of the acceptor stem region with a catalytic efficiency comparable to that of cleavage directed by the intact 5' half tRNA(Arg). The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNA(Arg) variants decreased. No heptamer-directed cleavage of a 3' half tRNA(Arg) was detected in the absence of T-stem base pairs. This strategy for targeted RNA cleavage using an RNA heptamer was applied to the cleavage of two target RNAs from HIV-1. An HIV-1 RNA containing a stable hairpin structure corresponding to the T stem-loop was cleaved efficiently, whereas no cleavage of a second HIV-1 target without any stable hairpin structure was detected. In this method, an RNA heptamer can direct efficient RNA cleavage with a higher specificity than expected.