A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]pyridinium) is a blue-absorbing molecular constituent of human ocular lipofuscin and contributes to the golden-yellow emission of this pigment. Lipofuscin photoproduces toxic reactive oxygen intermediates (ROI), but the specific molecular components responsible for this phototoxicity remain unidentified. In this article the aerobic photoreactivity of A2E is quantified by comparison with its biosynthetic precursor, all-trans-retinal, and with other appropriate standards. Under blue-light exposure the efficacies for formation of cholesterol (Ch) hydroperoxides and the superoxide radical anion (O2*-) were determined using high-pressure liquid chromatography with electrochemical detection and electron spin resonance oximetry and spin trapping, respectively. Photogeneration of singlet oxygen after blue-light excitation of A2E was demonstrated unambiguously by the Ch peroxidation assay. After blue-light irradiation of A2E, O2*- were detected, but the concentration was insufficient to account for the measured production of O2*- by the solvent extract of lipofuscin granules. The collective data support the conclusion that A2E does not produce sufficient concentrations of ROI to be the primary phototoxic constituent of lipofuscin.