A refocused primary electron spin-echo envelope modulation (RP ESEEM) technique and an adjustable frequency S/C-band pulsed EPR spectrometer have been used to produce ESEEM spectra with the lines due to nearby protons being greatly enhanced relative to those due to distant matrix protons. Application of this technique to the high pH (hpH) form of the Mo(V) center of sulfite oxidase has enabled nearby protons to be directly detected for the first time. Simulation of the RP ESEEM spectrum of the hpH form, suggests the presence of two nearby protons that have distributed hyperfine interactions (hfi); these protons are ascribed to a Mo-V-OH group with strong H-bonding interactions to other nearby proton donors or to the presence of a coordinated H2O ligand. The RP ESEEM technique promises to be widely applicable to the investigation of mutant forms of SO with altered Mo centers and paramagnetic centers in other metalloproteins where a nearby proton of interest is often masked by much more numerous distant protons and where high spectral resolution is not required. The distinctive differences in the CW and pulsed EPR spectra of the lpH and hpH forms are proposed to result from differences of the Mo-V-OH torsional angle and variations in the H-bonding interactions, which control the orientation of the Mo-V-OH proton(s) relative to the half-filled 4d(xy) orbital. The large isotropic hfi for the lpH form is suggested to result from an intramolecular MoV-OH ... S-cys hydrogen bonding interaction that places the proton of the Mo-V-OH group in the equatorial plane of the square pyramidal oxo-Mo(V) center of SO.