The three-dimensional ultrastructure of the filamentous glycocalyx of the brush border in the mouse small intestine was successfully demonstrated by high resolution scanning electron microscopy (SEM). The specimens were fixed with 2% glutaraldehyde in a 0.1 M phosphate buffer (pH7. 4), and rinsed with buffered solutions with differently adjusted pH values (pH3. 0, 7.0 or 11.0). They were then osmicated, dried, spatter-coated with gold (1.0-1.5 nm), and observed under a high resolution SEM. The glycocalyx on the luminal surface of the intestinal villi covered the top of the microvilli of the epithelial cells and were well preserved in the specimens treated with an alkaline buffer (pH11. 0). The glycocalyx was observed as filamentous structures, 7 to 15nm thick in diameter. These filaments repeatedly branched and anastomosed with neighboring ones to form an actual network or plexus as a whole, in contrast with superimposed images in transmission electron microscopy (TEM) which suggested that such anastomoses were pseudo-networks. The filaments thickened globularly at the sites of the filament bifurcation or branching. On the other hand, specimens rinsed with an acid or neutral buffer showed no glycocalyx on their microvilli, whose naked top had knob-like structures. Thus, the pH values of the washing buffer solutions were considered to affect the preservation of the surface coat due to molecular characteristics.