In photosystem II (PS II), chlorophyll, p-carotene, and cytochrome b(559) an alternate electron donors that may be involved in a photoprotection mechanism. The present study describes the use of high-field EPR spectroscopy to characterize the low-temperature photooxidation of Chit and Car cofactors in PS II. The EPR signals of the individual species, previously not resolved at X-band frequency (9 GHz), are resolved at higher D-band frequency (130 GHz) in deuterated Synechococcus lividus PS II. Deuteration of PS II results in significant narrowing of the EPR lines, yielding well-resolved EPR spectra of the Car(+) and Chl(Z)(+) radicals at 130 GHz. The g tensors of the individual species were determined by EPR spectral simulations. The g tensor determined for the Car(+) radical (g(xx) = 2.00335, g(yy) = 2.00251, g(zz) = 2.00227) is similar to that previously observed for a canthaxanthin cation radical but with a slightly rhombic tensor. The Chl(Z)(+) g tensor (g(xx) = 2.00312, g(yy) = 2.00263, g(zz) = 2.00202) is similar to that of a chlorophyll a cation radical. This study shows that both the carotenoid and chlorophyll radicals are generated in PS II by illumination at temperatures from 6 to 190 K and that there is no interconversion of Car(+) and Chl(Z)(+) radicals upon dark annealing at temperatures up to 160 K. This study also establishes the feasibility of using deuteration and high-field EPR to resolve previously unresolvable cofactor signals in PS II.