We have developed a novel method to study the interactions of nucleic acids with cationic species. The method, called phosphorus relaxation enhancement (PhoRE), uses H-1-detected P-31 NMR of exogenous probe ions to monitor changes in the equilibrium between free Mn2+ and Mn2+ bound to the RNA. To demonstrate the technique, we describe the interactions of four RNA molecules with metal ions (K+ and Mg2+), a small molecule drug (neomycin b), and a cationic peptide (RSG1.2). In each case, cationic ligand binding caused Mn2+ to be displaced from the RNA. Free Mn2+ was determined from its effect on the T-2 NMR relaxation rate of either phosphite (HPO32-) or methyl phosphite (MeOPH1 CH3OP(H)O-2(-)). Using this method, the effects of [RNA] as low as 1 muM could be measured in 20 min of accumulation using a low field (200 MHz) instrument without pulsed field gradients. Cation association behavior was sequence and [RNA] dependent. At low [K+], Mn2+ association with each of the RNAs decreased with increasing [K+] until similar to40 mM, where saturation was reached. While saturating K+ displaced all the bound Mn2+ from a 31-nucleotide poly-uridine (U-31), Mn2+ remained bound to each of three hairpin-forming sequences (A-site, RRE1, and RRE2), even at 150 mM K+. Bound Mn2+ was displaced from each of the hairpins by Mg2+, allowing determination of Mg2+ dissociation constants (K-d,K-Mg) ranging from 50 to 500 muM, depending on the RNA sequence and [K+]. Both neomycin b and RSG1.2 displaced Mn2+ upon binding the hairpins. At [RNA] similar to3 muM, RRE1 bound a single equivalent of RSG1.2, whereas neither RRE2 nor A-site bound the peptide. These behaviors were confirmed by fluorescence polarization using TAMRA-labeled peptide. At 2.7 muM RNA, the A-site hairpin bound a single neomycin b molecule. The selectivity of RSG1.2 binding was greatly diminished at higher [RNA]. Similarly, each hairpin bound multiple equivalents of neomycin at the higher [RNA]. These results demonstrate the utility of the PhoRE method for characterizing metal binding behaviors of nucleic acids and for studying RNA/ligand interactions.