Complementary techniques of chlorophyll a fluorescence, steady state CO(2) exchange, and O(2) release during a multiple turnover flash were applied to compare responses to irradiance for leaves of wild type and psbS mutants. The latter included variants in which the psbS gene was deleted (npq4-1) or possessed a single point mutation (npq4-9). Nonphotochemical quenching (NPQ) was reduced by up to 80 and 50%, respectively, in these lines at high irradiance. Analysis of changes in steady-state fluorescence yields and quantum yield of linear electron transport in the context of the reversible radical pair model of Photosystem II (PS II) indicated that NPQ occurs by nonradiative deactivation of chlorophyll singlet states in normal leaves. In contrast, application of the same criteria together with the observed irreversibility of NPQ and decline in density of functional PS II reaction centers following excessive illumination indicated a change in reaction center properties for the psbS deletion phenotype (Npq4-1(-)). Specifically, PS II reaction centers in Npq4-1(-) convert to a photochemically inactive, yet strongly quenching, form in intense light. The possibility of formation of a carotenoid or chlorophyll cation quencher in the reaction center is discussed. Results for the point mutant phenotype (Npq4-9(-)) were intermediate to those of wild-type and Npq4-1(-). Furthermore, wild-type leaves exhibited a significant reversible increase in the PS II in vivo rate constant for photochemistry (k(P0)) in saturating compared to limiting light. Changes in k(P0) could not be accounted for in terms of a classic phosphorylation-dependent (state transition) mechanism. Changes in k(P0) may arise from alternate pigment-protein conformations that alter the way excitons equilibrate among PS II chromophores. The lack of similar irradiance-dependent changes in k(P0) for the psbS mutants suggests a role for the PS II-S protein in the regulation of exciton distribution.