All-trans-retinal is the precursor of A2E, a fluorophore within lipofuscin, which accumulates in human retinal pigment epithelial (hRPE) cells and contributes to age-related macular degeneration. Here we have compared the in vitro dark cytotoxicity and visible-light-mediated photoreactivity of all-trans-retinal and A2E in hRPE cells. All-trans-retinal caused distinct cytotoxicity in hRPE cells measured with cell metabolic activity (MTS) and lactate dehydrogenase release assays. Significant increases in intracellular oxidized glutathione (GSSG), extracellular GSH and GSSG levels and lipid hydroperoxide production were observed in cells incubated in the dark with 25 and 50 μm all-trans-retinal. Light modified all-trans-retinal’s harmful action and decreased extracellular glutathione and hydroperoxide levels. A2E (<25 μm) did not affect cell metabolism or cytoplasmic membrane integrity in the dark or when irradiated. 25 μm A2E raised the intracellular GSSG level in hRPE cells to a much smaller extent than 25 μm all-trans-retinal. A2E did not induce glutathione efflux or hydroperoxide generation in the dark or after irradiation. These studies support our previous conclusions that although A2E may be harmful at high concentrations or when oxidized, its phototoxic properties are insignificant compared to those of all-trans-retinal. The endogenous production of A2E may serve as a protective mechanism to prevent damage to the retina by free all-trans-retinal.