Pulse EPR, 55Mn-ENDOR and ELDOR-detected NMR experiments were performed on the S2-state of the oxygen-evolving complex from spinach Photosystem II. The novel technique of random acquisition in ENDOR was used to suppress heating artefacts. Our data unambiguously shows that four Mn ions have significant hyperfine coupling constants. Numerical simulation of the 55Mn-ENDOR spectrum allowed the determination of the principal values of the hyperfine interaction tensors for all four Mn ions of the oxygen-evolving complex. The results of our 55Mn-ENDOR experiments are in good agreement with previously published data [Peloquin JM et al. (2000) J Am Chem Soc 122: 10926-10942]. For the first time ELDOR-detected NMR was applied to the S2-state and revealed a broad peak that can be simulated numerically with the same parameters that were used for the simulation of the 55Mn-ENDOR spectrum. This provides strong independent support for the assigned hyperfine parameters.