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 Mn2+, which provides a paramagnetic probe for EPR-based 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 Mn2+ site in the hammerhead ribozyme. Small changes in low-temperature X-band EPR signals are detected as signatures of Mn2+ ions in the RNA binding pocket. H-1 and P-31 Q-band ENDOR spectra are presented for Mn-hammerhead and Mn-nucleotide model complexes. The P-31 ENDOR data allow discrimination between direct Mn2+-phosphodiester coordination versus coordination through a hydrogen-bonded water molecule. Observation of hyperfine-coupled P-31 (A(P-31) similar to 4 MHz) provides evidence for direct coordination to a phosphodiester group in the hammerhead Mn2+ site. Exchangeable protons from aqueous ligands and nonexchangeable protons from base ligands also are examined for the Mn-nucleotide and Mn-ribozyme complexes. These signals indicate an ordered site for Mn2+ 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.