Metal ions are critical to the structure and function of many RNA molecules, but measuring detailed coordination environments in RNA is challenging under solution conditions. The phosphodiester bond cleavage reaction of the hammerhead ribozyme is activated by Mn 2+ , which provides a paramagnetic probe for EPRbased spectroscopic techniques. In this study, EPR and continuous-wave Q-band (34 GHz) electron nuclear double-resonance (ENDOR) spectroscopies have been used to investigate the coordination environment of a high-affinity Mn 2+ site in the hammerhead ribozyme. Small changes in low-temperature X-band EPR signals are detected as signatures of Mn 2+ ions in the RNA binding pocket. 1 H and 31 P Q-band ENDOR spectra are presented for Mn-hammerhead and Mn-nucleotide model complexes. The 31 P ENDOR data allow discrimination between direct Mn 2+ -phosphodiester coordination versus coordination through a hydrogenbonded water molecule. Observation of hyperfine-coupled 31 P (A( 31 P) ∼4 MHz) provides evidence for direct coordination to a phosphodiester group in the hammerhead Mn 2+ site. Exchangeable protons from aqueous ligands and nonexchangeable protons from base ligands also are examined for the Mn-nucleotide and Mnribozyme complexes. These signals indicate an ordered site for Mn 2+ in the hammerhead ribozyme and allow the ligand environment to be predicted, demonstrating the potential of ENDOR spectroscopy as a probe of RNA-metal interactions.