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Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.