A novel RNA recognition motif is characterized in an arginine-rich peptide. The motif, derived from lambda transcriptional antitermination protein N, regulates an RNA-directed genetic switch. Its characterization by multidimensional nuclear magnetic resonance (NMR) demonstrates specific RNA-dependent folding of N- and C-terminal recognition helices separated by a central bend. The biological importance of the bent alpha-helix is demonstrated by mutagenesis: binding is blocked by substitutions in the N peptide or its target (the boxB RNA hairpin) associated in vivo with loss of transcriptional antitermination activity. Although arginine side chains are essential, the peptide is also anchored to boxB by specific nonpolar contacts. An alanine in the N-terminal helix docks in the major groove of the RNA stem whereas a tryptophan in the C-terminal helix stacks against a purine in the RNA loop. At these positions all 19 possible amino acid substitutions have been constructed by peptide synthesis; each impairs binding to boxB. The pattern of allowed and disallowed substitutions is in accord with the results of random-cassette mutagenesis in vivo. The helix-bend-helix motif rationalizes genetic analysis of N-dependent transcriptional antitermination and extends the structural repertoire of arginine-rich domains observed among mammalian immunodeficiency viruses.