Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen ((1)O(2)*) in the intact and the Rieske-depleted cytochrome b(6)f complexes (Cyt b(6)f) from Bryopsis corticulans, as well as in the isolated Rieske Fe-S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b(6)f can be bleached by light-induced (1)O(2)*, and that the (1)O(2)* production can be promoted by D(2)O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, beta-carotene and glutathione. Under similar experimental conditions, (1)O(2)* was also detected in the Rieske-depleted Cyt b(6)f complex, but not in the isolated Rieske Fe-S protein. The results prove that Chl a cofactor, rather than Rieske Fe-S protein, is the specific site of (1)O(2)* formation, a conclusion which draws further support from the generation of (1)O(2)* with selective excitation of Chl a using monocolor red light.