PURPOSE To develop a new technique for detailed study of the spatial distribution of retinal and choroidal microvasculature and their relationship to neurons and glial cells at the cellular level in human cadaveric eyes. METHODS Twenty-six human donor eyes were used. Wherever possible, the central retinal artery and a branch of the posterior ciliary artery were individually cannulated and perfused with oxygenated Ringer's solution with 0.5% bovine serum albumin. The perfusion pressure was continuously monitored. Once residual blood was washed out, the perfusate solutions were switched to fixative, membrane-permeabilizing solution and selected labeling solutions. The eyes were then immersion fixed and the retina and choroid flat-mounted for immunolabeling and confocal imaging before cryosectioning. The microstructures of vascular, glial, and neuronal cells in the retina and the stroma in the choroid were studied. RESULTS The retinal microvasculature was fully perfused and stained by cannulation of the central retinal artery. Regional distribution of choroidal vasculature perfusion was dependent on the specific feeder artery cannulated. The detailed spatial relationship between endothelial cells, glial cells, and neurons at the cellular and subcellular levels was identified with confocal microscopy and immunohistochemical labeling of retinal sections. In the choroid, endothelial cells were clearly identifiable down to the level of the intracellular cytoarchitecture of the choriocapillaris, along with their relationship to Bruch's membrane and the feeding and drainage vessels. CONCLUSIONS A microperfusion fixation and staining technique has been developed that allows studies of the structural relationships of vascular, glial, and neuronal elements at the cellular level in human donor eyes.