Cell surface anionic sites on primary and transformed cultures of human vascular endothelium were studied using cationized ferritin (CF) as an ultrastructural marker. The native distribution of anionic sites on the upper (free) surfaces of cells, fixed in situ with glutaraldehyde, was uniform. Binding of the polycationic ligand, CF, in unfixed cells induced redistribution of anionic sites. Rapid formation of discrete patches of CF particles was followed by reappearance of binding between patches, movement of surface-bound CF into intercellular clefts, and endocytosis, over the next 30 min. Aldehyde-fixed cells, detached from the culture surface, bound CF on both upper and lower surfaces. The distribution and mobility of negatively charged membrane components in vascular endothelium may have relevance for thrombosis, atherogenesis, and vascular permeability.