During the first few enzymatic turnovers after dark-adaptation of photosystem II (PSII), the relaxation rate of the EPR signals from the Mn cluster and Y(D)(*) are significantly enhanced. This light-adaptation process has been suggested to involve the appearance of a new paramagnet on the PSII donor side [Peterson, S., Ahrling, K., Högblom, J., and Styring, S. (2003) Biochemistry 42, 2748-2758]. In the present study, a correlation is established between the observed relaxation enhancement and the redox state of the quinone pool. It is shown that the addition of quinol to dark-adapted PSII membrane fragments induces relaxation enhancement already after a single oxidation of the Mn, comparable to that observed after five oxidations in samples with quinones (PPBQ or DQ) added. The saturation behavior of Y(D)(*) revealed that with quinol added in the dark, a single flash was necessary for the relaxation enhancement to occur. The quinol-induced relaxation enhancement of PSII was also activated by illumination at 200 K. Whole thylakoids, with no artificial electron acceptor present but with an intact plastoquinone pool, displayed the same relaxation enhancement on the fifth flash as membrane fragments with exogenous quinones present. We conclude that (i) reduction of the quinone pool induces the relaxation enhancement of the PSII donor-side paramagnets, (ii) light is required for the quinol to effect the relaxation enhancement, and (iii) light-adaptation occurs in the intact thylakoid system, when the endogenous plastoquinone pool is gradually reduced by PSII turnover. It seems clear that a species on the PSII donor side is reduced by the quinol, to become a potent paramagnetic relaxer. On the basis of XANES reports, we suggest that this species may be the Mn ions not involved in the cyclic redox changes of the oxygen-evolving complex.