A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H+/e(-) -ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF1-gamma of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF1-gamma was only 36%, which indicates that in vivo the reduction of CF1-gamma could be limited by fast reoxidation and/or restricted accessibility of CF1-gamma to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.