The 11-subunit trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates transcription and translation by binding to several (G/U)AG repeats present in the trp leader and trpG transcripts. Filter binding assays were used to study interactions between L-tryptophan-activated TRAP and synthetic RNAs. RNAs that contained GAG and/or UAG repeats were tested while the length and sequence of the nucleotides separating adjacent trinucleotide repeats were altered. TRAP-RNA complexes formed with transcripts containing GAG repeats were more stable than those with transcripts containing UAG repeats or alternating GAG and UAG repeats. The stability of TRAP-RNA complexes also increased substantially when the number of GAG repeats was increased from five to six and from six to seven. A gradual increase in complex stability was observed when the number of GAG repeats was increased from 7 to 11. The optimal spacer between adjacent trinucleotide repeats was found to be 2 nucleotides, with A and U residues preferred over G and C residues. TRAP binding was specific for single-stranded RNA; TRAP could not bind to RNA containing GAG repeats base paired in a stable RNA duplex. Overall, our findings suggest that each L-tryptophan-activated TRAP subunit can bind one (G/U)AG repeat and that multiple TRAP subunit-RNA binding site interactions are required for stable TRAP-RNA association.