We report application of electron spin-echo envelope modulation (ESEEM) spectroscopy to the problem of metal coordination environments in structured RNA molecules. ESEEM has been used in conjunction with N-15-guanosine labeling to identify nitrogen ligation to a Mn2+ site in a hammerhead ribozyme and in Mn2+-model guanosine monophosphate (GMP) complexes. Hammerhead ribozyme complexes consisting of a 34-nucleotide RNA enzyme strand annealed to a 13-nucleotide DNA substrate strand were poised in 1 M NaCl as a 1:1 complex with Mn2+, conditions previously determined to populate a single high-affinity Mn2+ site (Horton, T. E.; Clardy, R. D.; DeRose, V. J. Biochemistry 1998, 51, 18094-18108). Significant modulation of the electron spin-echo from several low-frequency features is detected for the natural-abundance, N-14-hammerhead samples. At 3600 G, the main hammerhead three-pulse ESEEM features arise at 0.6, 1.9, 2.5, and 5.2 MHz and are nearly identical for a Mn2+-GMP complex under the same conditions. For a ribozyme having N-15-guanosine incorporated into the enzyme strand, as well as for an N-15-labeled Mn2+-GMP complex, the modulation is completely altered and consists of one main feature at 3.4 MHz and a smaller feature at the upsilon(n)(N-15) Larmor frequency of 1.6 MHz. Preliminary analysis of the ESEEM data reveals an apparent hyperfine coupling of A(N-14) similar to 2.3 MHz, similar to previously reported values for Mn2+ directly coordinated to histidine and imidazole. These data demonstrate the potential for ESEEM as a spectroscopic tool for metal ligand determination in structured RNA molecules.