Excess light energy absorbed by the chloroplast membranes of green plants is dissipated by nonradiative de-excitation in order to protect against photodamage. This is observed as the nonphotochemical quenching of chlorophyll fluorescence, which has been suggested to result from an alteration in the structure and function of the chlorophyll a/b light-harvesting complexes of photosystem II (LHCII) due to the combined effects of protonation and the de-epoxidation of bound violaxanthin to form zeaxanthin. In agreement with this hypothesis, it is shown that the light-harvesting chlorophyll a/b proteins purified from spinach leaves exhibit pH-stimulated quenching of chlorophyll fluorescence; this quenching shares all the key features observed for the nonphotochemical quenching of chlorophyll fluorescence in vivo. In the case of the two minor complexes, LHCIIa (CP29) and LHCIIc (CP26), quenching is much greater than in the bulk complex LHCIIb and is strongly inhibited by the reagent dicyclohexylcarbodiimide. The carotenoids violaxanthin and zeaxanthin cause strong inhibition and stimulation of quenching, respectively, in these complexes. The results of this study are consistent with the suggestion that the minor light-harvesting complexes play a crucial role in photoprotective energy dissipation in the photosynthetic membrane of green plants. Moreover, for the first time, a system using isolated LHCIIa and LHCIIc for the study of the regulation of light harvesting is described.