An inhibitor of plasminogen activator was purified to apparent homogeneity from human umbilical vein endothelial cell conditioned medium. The purification was achieved by a speedy and simple two-step procedure, without the use of denaturants. The purified protein was a single-chain glycoprotein with apparent molecular mass of 48 kDa. The purified inhibitor had a specific activity of 8500 U/mg protein and the activity could be stimulated about fourteenfold by treatment with denaturants. An antiserum to the purified inhibitor was raised in rabbits. It recognised plasminogen activator inhibitor from platelets and plasma as well as from cultured endothelial cells. The immunoglobulin fraction of the antiserum neutralised the functional activity of the inhibitor from all these sources.