The lectins Griffonia simplicifolia I and Lycopersicon esculentum were used to assess the presence of endothelium-specific glycoproteins in the microvasculature of the rat myocardium, diaphragm and superficial cerebral cortex. Organs fixed by intravascular perfusion were processed to obtain semithin (0.5 micron) and thin (less than 0.1 micron) frozen sections that were reacted with biotinylated lectin followed by streptavidin conjugated to Texas Red, for semithin sections, or by streptavidin conjugated to 5-nm colloidal gold particles, for thin sections. Lycopersicon esculentum lectin exclusively labeled the endothelium of all small vessels in all three microvascular beds; it did not bind to components of either the parenchyma or the extracellular matrix. Griffonia simplicifolia I lectin exclusively labeled the endothelium of the entire microvasculature in the myocardium and diaphragm, but marked primarily pericytes in the cerebral microvasculature. It did not label any parenchymal or interstitial organ component. At the electron microscope level, the lectin Griffonia simplicifolia I labeling was associated with the plasmalemma proper and especially with plasmalemmal vesicles and their introits, and Lycopersicon esculentum lectin bound primarily to the luminal plasmalemma in the microvascular beds of the myocardium and diaphragm. In the cerebral cortex, labeling of the microvasculature was clearly different: Griffonia simplicifolia I bound primarily to pericytes and vascular smooth muscle cells whereas Lycopersicon esculentum labeled only the microvascular endothelium. Lysates prepared from the myocardium, diaphragm and cerebral cortex were processed through Griffonia simplicifolia I lectin affinity separation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fraction obtained. A number of putative endothelium-specific glycoproteins was detected and found to differ qualitatively and quantitatively from organ to organ. The most prominent polypeptide, approximately 97 kDa, was present in substantial amounts in the myocardium and diaphragm, but in considerably lower concentration in the cerebral cortex. The reverse applied for a approximately 55 kDa protein. The preferential distribution of the approximately 97 kDa protein parallels differences in Griffonia simplicifolia I lectin binding by fluorescence and electron microscopy on sections of the corresponding organs. The results provide further evidence for the existence of endothelial glycoproteins specific for different microvascular beds and possibly connected with local functional differentiations.