A small RNA ligand with high affinity for the HIV-1 Rev protein, generated by the SELEX in vitro evolution method, was used in a series of chemical modification studies to aid in determining the secondary structure of the ligand, to detect which modifications interfere with the binding of the ligand to Rev, and to find those modifiable groups that are protected from attack when bound to the Rev protein. This SELEX RNA ligand, like the high-affinity binding site of the Rev-responsive element, seems to bind the Rev protein within or along the major groove. There are two major regions of the RNA that interact with the Rev protein, and these regions appear to be close in space. Additionally, this high-affinity ligand has been used as the basis for an additional "biased randomization" SELEX procedure, in an effort to gain comprehensive information on the RNA sequences and structural elements necessary for efficient binding to the Rev protein. This complementary experimental approach supports the structural conclusions of our chemical modification data.