A quick identification of a cleavage site in the target RNA molecule to obtain sequence-specific cleavage by either catalytic RNA (ribozymes) or DNA (DNA enzymes) is very important for achieving gene-specific suppression. These molecules could also provide important information on the secondary and tertiary structure of the target RNA molecule. We have exploited the use of two kinds of DNA enzymes, namely, the 10-23 and 8-17 catalytic motif containing DNA enzymes, to achieve these objectives. We identified several DNA enzyme cleavage sites in the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) RNA-a structural feature present at the 5' end of all HIV-1 transcripts. Most of the DNA enzymes that cleaved the TAR RNA were targeted to the regions that were single-stranded in the predicted structure. Regions that were predicted to be base-paired (stem) failed to show any detectable cleavage. The DNA enzyme possessing the 8-17 catalytic motif was extremely efficient in cleaving full length, as well as short, HIV-1 specific transcripts. The efficiency of cleavage of the same target RNA by DNA enzymes that possessed the 10-23 catalytic motif was significantly less in comparison, and they failed to cleave the short transcripts. These molecules, in principle, have the potential to down regulate expression of all HIV-1 transcripts from a wide range of isolates because this region is functionally very well conserved.