Electron paramagnetic resonance (EPR) spectroscopy was utilized to investigate the correlation between RNA structure and RNA internal dynamics in complexes of HIV-1 TAR RNA with small molecules. TAR RNAs containing single nitroxide spin-labels in the 2′-position of U23, U25, U38, or U40 were incubated with compounds known to inhibit TAR-Tat complex formation. The combined changes in nucleotide mobility at all four sites, as monitored by their EPR spectral width, yield a dynamic signature for each compound. The multicyclic dyes Hoechst 33258, DAPI, and berenil bind to TAR RNA in a similar manner and gave nearly identical signatures. Different signatures were obtained for the acridine derivative CGP 40336A and the aminoglycoside antibiotic neomycin, which bind to different regions of the RNA. The dynamic signature for guanidinoneomycin was remarkably similar to that obtained for argininamide and is evidence for guanidinoneomycin binding to the same site as arginine 52 of the Tat protein, rather than to the neomycin binding site. The data presented here show that the dynamic signatures provide strong insights into RNA structure and recognition and demonstrate the value of EPR spectroscopy for the investigation of small molecule binding to RNA.