Strong inhibition of uncoupled photosynthetic electron transport by Cu(2+) in isolated spinach chloroplasts was observed by measuring changes in O(2) concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu(2+) to the chloroplast membranes resulted in removal of Cu(2+) from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu(2+) binding to inhibitory sites. Preincubation in the dark resulted in Cu(2+) binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.When the photosystems were assayed separately, photosystem I was more resistant to inhibition than photosystem II. The most sensitive site to the inhibitor was the oxidizing side of photosystem II.