It has recently been demonstrated that strong illumination under anaerobic conditions leads to the double reduction of the primary quinone acceptor, QA, which in turn promotes the light-induced formation of triplet reaction center chlorophyll, 3P680, a potentially dangerous species to its protein surroundings in the presence of oxygen [Vass, I., Styring, S., Hundal, T., Koivuniemi, A., Aro, E.-M., & Anderson, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1408-1412]. Here we have studied in further detail the formation of 3P680 producing centers in anaerobically photoinhibited photosystem II membranes by using low-temperature EPR spectroscopy. The results show that 3P680 formation occurs in three different populations of modified photosystem II centers. After a short period of photoinhibitory illumination, a very stable form of singly reduced QA is observed, with a decay halftime of several minutes at room temperature, and our results indicate that already this population of centers promotes the light-induced formation of the spin-polarized EPR signal from 3P680. The formation of these centers is enhanced below pH 6.0, indicating the involvement of a protonation event in neutralizing the negative charge on QA-, a prerequisite for efficient primary charge separation and subsequent triplet formation via the radical pair mechanism. If these centers are incubated in the dark, the stable singly reduced QA species is slowly reoxidized concomitant with the loss of its triplet forming ability. Extended photoinhibitory illumination converts the stable form of singly reduced QA to an EPR-silent species indicating the second reduction of QA-. The second negative charge on the double-reduced QA is neutralized most likely by a second protonation.(ABSTRACT TRUNCATED AT 250 WORDS)