Abstract The 505 nm change in isolated chloroplasts of Lactuca sativa var. Manoa showed a linear correlation with violaxanthin decrease. The corresponding difference extinction coefficient was established as 27.3 mM −1 · cm −1. The 505 nm change followed first-order kinetics. The first-order rate constant, a measure of de-epoxidase activity, and the final extent of the 505 nm change, a measure of the extent of de-epoxidation, varied with light intensity. In 670 nm light the activity remained about half-maximal at intensities from 1 to 4 kerg · cm −2 · s −1 before reaching saturation at 9 kergs · cm −2 · s −1. In contrast, the extent showed a simpler response and reached saturation already at 4 kergs · cm −2 · s −1. These results suggest that different photosynthetic factors determine de-epoxidase activity and de-epoxidation extent. De-epoxidase activity is known to reflect steady-state thylakoid pH whereas the de-epoxidation extent was found to reflect light-dependent variable substrate availability. The available fraction varied in size from 40% of the total violaxanthin at 1 kerg · cm −2 · s −1 to 67% at saturation. The possibility that variable availability reflects conformational changes of the membrane near Photosystem II which result in variable exposure of the substrate to enzyme is discussed.