Recent data from various vascular beds suggest that a layer of mucopolysaccharides covering the endothelial cells play an important role in transport processes, among others. In this study, electron microscopy (EM) was used to explore the presence of an endothelial surface layer (ESL) in rat glomerular capillaries. We adopted various fixation and labeling techniques, as follows: (1) negatively charged lipid particles were used as a tracer that was expected to be excluded from the ESL. The density of intravascular lipid particles in flow-arrested capillaries was 89% lower in a 200-nm periendothelial area than in the rest of the luminal space (n = 6 rats, P < 0.001); (2) podocytes of cryofixed fresh tissue had a 20-nm extramembranous coat, interpreted as the true glycocalyx; the coat was less expressed on the endothelium; (3) on unfixed endothelial cells, colloidal lanthanum labeled a 60-nm-thick layer, occasionally forming lumps; (4) perfusion with a fluorocarbon-based oxygen-carrying fixative, followed by tannic acid contrast enhancement, revealed an extensive (> 200 nm) ESL not previously described; however, this finding was restricted to superficial glomerular capillaries; (5) Cupromeronic Blue cytochemistry displayed a loose proteoglycan network in fenestral openings and, occasionally, a semiordered ESL; (6) ferricyanide-reduced osmication resulted in increased numbers of fenestral diaphragms. In conclusion, this study provides novel morphological evidence to support the presence of a significant glomerular ESL.