The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.