Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H(+) accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Delta[Formula: see text] (H+)) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca(2+) binding to the lumen side of the thylakoid membrane; in particular, Ca(2+) binding to the 8 kDA subunit III of the CF(o) H(+) channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca(2+) from the CF(o) H(+) channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation.