The human immunodeficiency virus Tat protein binds specifically to an RNA stem-loop structure (TAR) that contains two helical stem regions separated by a three-nucleotide bulge. A single arginine within the basic region of Tat mediates specific binding to TAR, and arginine as the free amino acid also binds specifically to TAR. We have previously proposed a model in which interaction of the arginine guanidinium group with guanosine-26 (G26) and with a pair of phosphates is stabilized by formation of a base triple between U23 in the bulge and A27.U38 in the upper helix. Here we show by NMR spectroscopy that formation of the base triple is critical for arginine binding to TAR. Mutants of TAR that cannot form the base triple or that remove the guanine contact do not bind arginine specifically. These mutants also showed reduced transactivation by Tat. A triple mutant designed to form an isomorphous base triple between C23 and G27.C38 binds arginine and adopts the same conformation as wild-type TAR. These results demonstrate the importance of RNA structure for arginine binding and further demonstrate the direct correspondence between arginine and Tat binding.