The mobility and distribution of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 220.127.116.11) and a specific endothelial cell surface protein was assessed by fluorescein-conjugated monoclonal antibodies on bovine and murine endothelial cells grown on their extracellular matrix. The combination of data obtained from fluorescence recovery after photobleaching measurements and observations under epifluorescence and total internal reflection fluorescence reveals a restriction of these protein markers to the apical membrane of endothelial cell. This asymmetry is evident both when cells are grown at a sparse density or at confluence. When cells are brought into suspension, the fluorescein-conjugated antibody is found over the entire cell surface. The fluorescence disappears from the basal part of the cell when the cells are again spread on coverslips coated with a layer of extracellular matrix. Conversely, cells spread on glass coverslips without extracellular matrix do not show this restriction phenomenon. It is suggested that the extracellular matrix provides the signal to induce the restricted topology of membrane protein markers on endothelial cells.