Bisretinoids are a family of fluorophores that form in photoreceptor cells' outer segments by nonenzymatic reaction of two vitamin A aldehydes (A2) with phosphatidylethanolamine (PE). Bisretinoid fluorophores are the major constituents of the lipofuscin of retinal pigment epithelium (RPE) that accumulate with age and contribute to some retinal diseases. Here, we report the identification of a previously unknown fluorescent bisretinoid. By ultra-performance LC (UPLC) coupled to photodiode array detection, fluorescence (FLR), and ESI-MS, we determined that this novel bisretinoid is 1-octadecyl-2-lyso-sn-glycero A2PE (alkyl ether lysoA2PE). This structural assignment was based on molecular mass (m/z 998), UV-visible absorbance maxima (340 and 440 nm), and retention time (73 min) and was corroborated by biomimetic synthesis using all-trans-retinal and glycerophosphoethanolamine analogs as starting materials. UPLC profiles of ocular extracts acquired from human donor eyes revealed that alkyl ether lysoA2PE was detectable in RPE, but not neural retina. LysoA2PE FLR spectra exhibited a significant hyperchromic shift in hydrophobic environments. The propensity for lysoA2PE to undergo photooxidation/degradation was less pronounced than A2E. In mechanistic studies, A2PE was hydrolyzed by phospholipase A2 and plasmalogen lysoA2PE was cleaved under acidic conditions. The characterization of these additional members of the bisretinoid family advances our understanding of the mechanisms underlying bisretinoid biogenesis.