Pulsed electron paramagnetic resonance at the microwave K-a band (similar to 30 GHz) was used to study the coordination of adenosine nucleotides to Mn2+ at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electron-nuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques. are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mn nucleotide complexes. Therefore, to directly detect binding of the Mn nucleotide to myosin, we used non-hydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electron-electron double resonance to detect spin probe manganese dipolar interaction. We found that under substoichiometric conditions, both Mn.AMPPNP and Mn.ADP.AlF4 form a complex with myosin, and Mn.ADP does not form such a complex. This correlates well with the biological dissociation of Mg.ADP from myosin after the hydrolysis of ATP. The analysis of P-31 ENDOR spectra reveals that in Mn.AMPPNP, Mn.ATP, and Mn.ADP at myosin or in solution, the nucleotide is coordinated to Mn2+ by two phosphate groups, whereas in Mn.ADP.AlF4, only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of Mn.ADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ion's is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metal-protein binding when such binding does not provide reliable spectroscopic signatures.