Diapedesis of leukocytes across endothelial barriers is generally believed to require the opening of endothelial tight junctions. At the blood-brain barrier (BBB), endothelial cells are interconnected by complex tight junctions. Here, we show by serial section conventional electron microscopy that during experimental autoimmune encephalomyelitis mononuclear cells traverse cerebral microvessels by a transcellular pathway, leaving the endothelial tight junctions intact. Cerebral endothelial cells were found to form filopodia-like membrane protrusions on their luminal aspect, thus embracing the mononuclear cells and forming cup-like structures, and eventually pores, through which the traversing cell could reach the abluminal side. At the abluminal side endothelial cell protrusions surrounding a migrating inflammatory cell were found to be progressively lined with basal lamina, suggesting a change from luminal to abluminal membrane characteristics of endothelial cell membranes during inflammatory cell diapedesis. Morphological evidence for the involvement of tight junctions in the diapedesis of mononuclear cells across the inflamed BBB could not be obtained in any case. Taken together, the presence of morphologically intact tight junctions and our novel finding of the presence of a basal lamina on both sides of abluminal endothelial cell protrusions surrounding migrating inflammatory cells suggests that during experimental autoimmune encephalomyelitis diapedesis of mononuclear cells occurs via a transendothelial process.