A single arginine residue within the RNA-binding domain of the human immunodeficiency virus Tat protein makes a critical sequence-specific contact to TAR RNA. Arginine as the free amino acid also binds specifically to TAR and induces a change in RNA conformation similar to that induced by Tat peptides. NMR and biochemical studies have suggested that the arginine-binding site is stabilized by a base triple interaction between a bulged U and an A x U base pair in the adjacent stem. In this study, we have used chemical modification and mutagenesis experiments to examine the relative contributions of the Watson-Crick and Hoogsteen base-pairing partners of the proposed U-A x U base triple. We show that the Hoogsteen interaction is critical for arginine binding whereas the Watson-Crick interaction can be eliminated or replaced by other base-base interactions. The results are consistent with biochemical studies of the Tat-TAR interaction and support the base triple model for the structure of TAR.