We have developed a new method to quantify the transmembrane electrochemical proton gradient present in chloroplasts of dark-adapted leaves. When a leaf is illuminated by a short pulse of intense light, we observed that the light-induced membrane potential changes, measured by the difference of absorption (520 nm-546 nm), reach a maximum value (approximately 190 mV) determined by ion leaks that occur above a threshold level of the electrochemical proton gradient. After the light-pulse, the decay of the membrane potential follows a multiphasic kinetics. A marked slowdown of the rate of membrane potential decay occurs approximately 100 ms after the light-pulse, which has been previously interpreted as reflecting the switch from an activated to an inactivated state of the ATP synthase (Junge, W., Rumberg, B. and Schröder, H., Eur. J. Biochem. 14 (1970) 575-581). This transition occurs at approximately 110 mV, thereby providing a second reference level. On this basis, we have estimated the Delta micro (H(+)) level that pre-exists in the dark. Depending upon the physiological state of the leaf, this level varies from 40 to 70 mV. In the dark, the Delta micro (H(+)) collapses upon addition of inhibitors of the respiratory chain, thus showing that it results from the hydrolysis of ATP of mitochondrial origin. Illumination of the leaf for a period longer than several seconds induces a long-lived Delta micro (H(+)) increase (up to approximately 150 mV) that reflects the light-induced increase in ATP concentration. Following the illumination, Delta micro (H(+)) relaxes to its dark-adapted value according a multiphasic kinetics that is completed in more than 1 h. In mature leaf, the deactivation of the Benson-Calvin cycle follows similar kinetics as Delta micro (H(+)) decay, showing that its state of activation is mainly controlled by ATP concentration.