Purpose: Lipids represent 50% of retinal dry matter. Within structural phospholipids, ether-lipids are a major class but their exact species, concentrations and functions in the retina are still unknown. A defect in their biosynthesis is observed in peroximal disorders like Rhizomelic Chondrodysplasia Punctata type 2 and 3 (RCDP) characterized by retinal pigment epithelium (RPE) degeneration and defects in ocular vasculogenesis. The aim of this work was to perform a detailed study of the retinal composition in ether-lipids. Methods: Total lipids were purified from calf retina and RPE samples. The proportion of major lipid classes was determined by thin-layer chromatography (TLC). Phospholipids were separated from non-phosphorus lipids by liquid chromatography (LC), subclasses (linked with ethanolamine, choline, serine or inositol) were quantified by LC and then purified by TLC. The fatty acid composition of each subclass was evaluated by gas chromatography (GC). The expression of the key enzyme of ether-lipids biosynthesis (DHAP-AT) was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR) on calf retina and RPE samples and its cellular localization was performed on rat retina semi-thin sections by in situ hybridization. Results: Phospholipids represent 85% of retinal and RPE lipids. Within phospholipids 41% and 36% were linked with ethanolamine in the retina and the RPE, respectively. Ether-lipids accounted for 30% of ethanolamine-linked phospholipids in both the retina and the RPE. In the retina, 67% of ethanolamine ether-lipids contained stearic acid in position 1 of glycerol, and 15% contained palmitic acid. Stearic acid accounted for 38% and palmitic acid for 41% in the RPE. DHAP-AT was expressed in both, retinal and RPE cells and its expression within the retina was mainly localized in the photoreceptor inner segments and in bipolar cells to a lesser extent. Conclusions: Our results illustrate the quantitative importance of ether-lipids and particularly those linked to ethanolamine in retinal and RPE cells. According to these results and considering their putative structural and metabolic roles in other tissues, ether-lipids may play a crucial role in retinal physiology as illustrated by their lack in RCDP patients.