The luminal surface of endothelial cells is lined with the glycocalyx, a network structure of glycoproteins probably 50 to 100 nm thick. It has been suggested that a relatively regular fibre-matrix structure may be responsible for the ultrafiltration properties of microvascular walls, both when the endothelium is continuous and when it is fenestrated. Positive structural evidence demonstrating an underlying periodicity in the glycocalyx has been hard to obtain. Here we present structural analysis of glycocalyx samples prepared in a variety of ways for electron microscopy. Using computed autocorrelation functions and Fourier transforms of representative areas of the electron micrograph images, we show that there is an underlying three-dimensional fibrous meshwork within the glycocalyx with characteristic spacings of about 20 nm. Together with a fibre diameter consistent with our observations of about 10-12 nm, the 20-nm spacing provides just the size regime to account satisfactorily for the observed molecular filtering; the observations are consistent with the fibre matrix model. We also show that the fibrous elements may occur in clusters with a common intercluster spacing of about 100 nm and speculate that this may reveal organisation of the glycocalyx by a quasi-regular submembranous cytoskeletal scaffold.