The plasminogen activator activity (PAA) in extracts of the intima, media, and adventitia of the normal human aorta and other large arteries (carotid artery, renal artery and iliac artery) was studied with a sensitive, quantitative spectrophotometric assay using plasminogen and the chromogenic plasmin substrate S-2251. All layers of the arteries showed PAA which was highest in the adventitia, lowest in the media, while in the intima (aorta) PAA was intermediate, but much closer to that of the media. Plasminogen activator inhibition (PAI) was at the same level in all layers of the arteries studied. Plasmin inhibition (PI) was higher in adventitia than in intima (aorta), while in media the PI was intermediate. The PAA was due to the tissue-type plasminogen activator (t-PA), but not to the urokinase-type (u-PA), as judged by addition of respective antibodies. The relatively low PAA found in the intima of large arteries is therefore due to a low plasminogen activator and not a high plasminogen activator inhibitor activity or plasmin inhibitor level.