Lung macro- and microvascular endothelial cells exhibit unique functional attributes, including signal transduction and barrier properties. We therefore sought to identify structural and functional features of endothelial cells that discriminate their phenotypes in the fully differentiated lung. Rat lung macro- (PAEC) and microvascular (PMVEC) endothelial cells each exhibited expression of typical markers. Screening for reactivity with nine different lectins revealed that Glycine max and Griffonia (Bandeiraea) simplicifolia preferentially bound microvascular endothelia whereas Helix pomatia preferentially bound macrovascular endothelia. Apposition between the apical plasmalemma and endoplasmic reticulum was closer in PAECs (8 nm) than in PMVECs (87 nm), implicating this coupling distance in the larger store operated calcium entry responses observed in macrovascular cells. PMVECs exhibited a faster growth rate than did PAECs and, once a growth program was initiated by serum, PMVECs sustained growth in the absence of serum. Thus, PAECs and PMVECs differ in their structure and function, even under similar environmental conditions.