An ultrastructural study was done on puromycin aminonucleoside (PAN) nephropathy which was induced in a group of Sprague-Dawley rats by a single intraperitoneally injected dose. To study the ultrastructural alteration of glomerular anionic sites renal tissue was stained with polyethyleneimine (PEI) as a cationic probe. The PEI method seemed to selectively stain heparan sulfate proteoglycan in the basement membrane and has been widely used to evaluate the changes of the basement membrane in human diseases as well as in experimental work. The experimental rats developed proteinuria three days after the PAN injection. Electron microscopic studies of glomeruli showed the loss of epithelial foot processes, formation of cytoplasmic vacuoles, microvillous formation, and increased numbers of lysosomes in the cytoplasm of podocytes. The anionic sites on the basement membrane with foot process fusion were mostly indistinguishable from those seen in control rats, but focal areas of loss or disarray of anionic sites were noted. The anionic sites were not seen on the basement membrane where the overlying epithelium was detached. The results suggest that proteinuria in PAN nephrosis may be primarily due to a glomerular epithelial lesion, leading to focal disarray of anionic sites or focal defects in the epithelial covering of the basement membrane. The loss of anionic sites in the basement membrane may result partially from the foot process fusion, but mostly from the epithelial detachment.