Endothelial cells were isolated from arteries and veins obtained from elderly people at autopsy and propagated for 37 to 69 population doublings. The cells secreted tissue-type plasminogen activator (t-PA) and PA inhibitor-1, and, after subculturing, urokinase-type PA (u-PA) antigen. The following differences between endothelial cells from adult arteries and veins were observed: 1) The cells had the potential to be propagated as a healthy monolayer. The diameter of aortic endothelial cells increased after 8 to 19 population doublings, while a homogeneous population of small diameter vena cava cells was retained for 35 population doublings. 2) The amount of secreted t-PA varied. Vena cava cells produced four times more t-PA than aorta cells, and 20-fold more than umbilical artery or vein endothelial cells. The t-PA mRNA content of vena cava cells did not exceed that of aorta cells, but was fourfold greater than that of umbilical cord endothelial cells. 3) The release of u-PA antigen varied. No u-PA antigen was detectable in conditioned medium of primary cultures of human aorta and vena cava endothelial cells or of early passage vena cava cells. After prolonged subculturing, vena cava cells started to secrete u-PA. Endothelial cells from aorta and other adult arteries, however, started secreting u-PA after one to four passages, parallel to the occurrence of enlarged endothelial cells. u-PA was present as a u-PA/inhibitor complex and as a single-chain u-PA. These differences may be developmentally related to their artery or vein origin or may reflect differences acquired during the "life history" of these blood vessels in vivo. Our data suggest that the release of u-PA antigen by human macrovascular endothelial cells can be used as an indicator of cell senescence.