Exposure of isolated intact mitochondria to near UV to visible light resulted in not only loss of respiration, the most well-documented phenomenon regarding phototoxic effects in the respiring organelles, but also lipid peroxidation of membranes and mitochondrial swelling; these turned out to be O$_2$-dependent and thus prevented by anaerobiosis, enhanced by a partial deuteration of the suspension medium, and suppressed by the presence of a singlet oxygen ($^1O_2$) scavenger. Measurements of the spectral dependence of such detrimental effects of light on mitochondrial structure and function revealed that all the resulting spectra bear a significant resemblance to the action spectrum for photogeneration of $^1O_2$ from mitochondrial membranes, which in turn carries the spectral characteristics of light absorption by mitochondrial Fe-S centers. Futhermore, destructing the Fe-S centers by a mercurial treatment of mitochondria brought about a striking reduction of the light-induced membrane peroxidation and swelling of mitochondria. These results are consistent with the suggestion that the impairment of functional, structural integrity of mitochondria caused by strong irradiation is directly related to the production of $^1O_2$ in mitochondria, photosensitized by the Fe-S centers. This paper also presents kinetic data which indicate that, among various membrane-bound protein systems associated with mitochondrial energy metabolism, the respiratory chain is the primary target for photodamage.