Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe associated with the quinones Q and Q in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9 in 2-propanol and Q in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9 in vitro and on Q in the iron-depleted PS II samples. For the former a complete set of isotropic H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the α-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. H-ENDOR spectra of Q in iron-depleted PS II samples have been measured in buffers made in HO and DO. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of Q and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.