Several random mutations have been generated in the psbA2 gene of Synechocystis sp. PCC 6803 [Narusaka, Y., Murakami, A., Saeki, J., Kobayashi, H., and Satoh, K. (1996) Plant Sci. 115, 261-266]. The phototolerant mutant (I6) carrying all the amino acid substitutions in the lumenal side of D1 protein (S322I, I326F, and F328S) and a site-directed mutant of the same phenotype (NDFS) substituted in the stromal side of the protein (N234D and F260S) were characterized by thermoluminescence measurements. We observed (1) no significant differences in their growth rates at either low or high light irradiance, (2) a downshifted B-band in the NDFS mutant, (3) an upshifted Q-band in the I6 mutant, and (4) a damped period four oscillation of thermoluminescence in the B-band of both mutants. By examining the possible implications of these results on the redox properties of the PS II components in the mutants, we concluded that equilibrium constants for sharing an electron between the primary (QA) and secondary acceptor plastoquinones (QB) are decreased in both mutants.