We devised a single-step selection method to identify short sequences within the folded Rev response element (RRE) of the human immunodeficiency virus (HIV) RNA genome that are proximal and able to bind short oligonucleotides. The method employed a library of partially randomized tethered oligonucleotide probes (TOPs) complementary to all regions of the RRE and an RNase H cleavage assay which identified those RRE regions preferred by the TOPs. Six short sequences were identified. Two TOPs synthesized on the basis of this selection, S1-5-S2 and S1-1-S2, were potent, concentration-dependent inhibitors of a RRE function in vitro and abolished interaction of the RRE with the HIV regulatory protein Rev at nanomolar concentration (25-degrees-C, 0.8 nM RRE, 45 nM Rev). TOPs S1-5-S2 and S1-1-S2 exhibited greater potency and faster association kinetics than traditional oligonucleotides targeted to the same regions. The random TOP selection procedure described here allows rational design of oligonucleotide-based ligands for RNA that exploit (rather than avoid) the complex structure of the RNA target.