The autofluorescent lipofuscin that accumulates in retinal pigment epithelial cells with age may contribute to an age-related decline in cell function. The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff base, N-retinylidene phosphatidylethanolamine from all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane; (ii) further reaction of N-retinylidene phosphatidylethanolamine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (iii) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogenic scheme, all-trans-retinal was reacted with dipalmitoyl-L-α-phosphatidylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass spectrometry revealing the molecular ion at m/z 1222.9 (C77H124O8PN) accompanied by production at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromatography peaks, each with UV similar to reference A2-PE. By mass spectrometry, A2-PE consisted of multiple peaks, representing fatty acids with different chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-PE as the A2E precursor.