Accumulation of lipofuscin (LF) is a prominent feature of aging in the human retinal pigment epithelium (RPE) cells. This age pigment exhibits substantial photoreactivity, which may increase the risk of retinal photodamage and contribute to age-related maculopathy. In a previous study, we detected singlet oxygen generation by lipofuscin granules excited with blue light. In this paper we investigated the ability of hydrophobic components of lipofuscin to photogenerate singlet oxygen in non-polar environments. Singlet oxygen was detected directly by monitoring its characteristic phosphorescence at ca 1270 nm. The action spectrum of singlet oxygen formation indicated that this process was strongly wavelength-dependent and its efficiency decreased with increasing wavelength by a factor of ten, comparing 420 nm and 520 nm. The quantum yield of singlet oxygen increased with increasing concentration of oxygen. Using laser flash photolysis we studied the possible mechanism of singlet oxygen formation. The observed transient, with a broad absorption spectrum peaking at around 440 nm, was identified as a triplet with lifetime ca 11 microseconds. It was quenched by both molecular oxygen and beta-carotene with concomitant formation of a beta-carotene triplet state. These results indicate the potential role of hydrophobic components of lipofuscin in blue light-induced damage to the RPE.