We recently developed a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry (IMS) system. This system enables us to perform structural analyses using tandem mass spectrometry (MS/MS), as well as to visualize phospholipids and peptides in frozen sections. In the retina, phototransduction is regulated by the light-sensitive interaction between visual pigment-coupled receptor proteins, such as rhodopsin, and G proteins, such as transducin. There are some reports that the conformation of rhodopsin is influenced by the composition of phospholipids in the lipid bilayer membrane. However, these results were based on in vitro experiments and have not been analyzed in vivo. In this study, we visualized and identified phospholipids in mouse retinal sections with the MALDI-QIT-TOF-based IMS system. From a spectrum obtained by raster-scanned analysis of the sections, ions with high signal intensities were selected and analyzed by MS/MS. As a result, sixteen ions were identified as being from four diacyl-phosphatidylcholine (PC) species, i.e., PC (16:0/16:0), PC (16:0/18:1), PC (16:0/22:6), and PC (18:0/22:6), with different ion forms. The ion images revealed different distributions on the retinal sections: PC (16:0/18:1) was distributed in the inner nuclear layer and outer plexiform layer, PC (16:0/16:0) in the outer nuclear layer and inner segment, and both PC (16:0/22:6) and PC (18:0/22:6) in the outer segment and pigment epithelium. In conclusion, our in vivo IMS analyses demonstrated a three-zone distribution of PC species on the retinal sections. This approach may be useful for analyzing lipid changes and their contribution to phototransduction in the retina. Copyright © 2008 John Wiley & Sons, Ltd.