Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R 9 ) also bound specifically to TAR, and a mutant Tat protein that contained R 9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K 9 ) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K 9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine η nitrogens and the ε nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures.