Intrinsic differences between aortic and venous endothelial cells were demonstrated by studies of morphology, growth rates, and protein synthesis. Endothelial cells from bovine thoracic aortas (AECs) and inferior vena caval (VECs) were harvested, maintained in cell culture, and characterized. VECs were consistently larger (mean cell diameter 14.3 micron vs 12.7 micron; p less than 0.001) and more pleomorphic than were AECs. Scanning electron microscopy revealed that VECs were thinner than AECs and that VECs had numerous fine, cellular processes that were much less abundant on the AECs. Enzymatic disaggregation of confluent cells resulted in a reduced generation time by the AECs compared with VECs. Moreover, VECs responded to disaggregation by a significantly larger increase in cell size than the AECs. Protein synthesis was quantitated by computer analysis of autoradiograph of two-dimensional gel eletrophoresis and separation of 998 35S-methionine-labeled proteins from cell lysates. Synthesis of 257 proteins was significantly different (p less than 0.05); synthesis of 239 proteins by VECs was either decreased (219) or undetectable (20) compared with those of AECs. In contrast, only 18 proteins were produced in significantly greater quantity by VECs than by AECs. The cytoskeletal proteins actin and alpha- and beta-tubulins were produced in significantly greater quantity by AECs than VECs. These results indicate that cultured endothelial cells of arterial origin are substantially different from those of the venous circulation. These phenotypic differences are maintained in vitro despite eliminating variability in hemodynamic stress and interaction with other cellular elements of the vessel wall.