Intact or broken chloroplasts exhibited strong reversible quenching of chlorophyll fluorescence when illuminated with red light in the absence of oxygen and other electron acceptors. Fluorescence quenching was not accompanied by formation of a significant proton gradient. It was caused by excitation of photosystem II, not photosystem I. The extent of quenching and its quantum yield was increased by carbonyl cyanide-m-chlorophenylhydrazone (CCCP), which protected chloroplasts against photobleaching of pigments in the absence of oxygen. CCCP also increased reversibility of fluorescence quenching, perhaps by facilitating cyclic electron transfer around photosystem II. Addition of oxygen or, in intact chloroplasts, of nitrite or oxaloacetate, increased fluorescence. A similar increase was brought about by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or other inhibitors that interrupt electron flow between the photosystems. In the presence of these inhibitors, fluorescence quenching could no longer be observed but it was restored by the addition of dithionite. It is proposed that a main part of anerobic fluorescence quenching is caused by accumulation of reduced phaeophytin, and that this accumulation is prevented by electron transport inhibitors acting between photosystem II and I.