Conditioned serum-free medium both from confluent porcine aortic endothelial cells and from human venous and arterial umbilical cord endothelial cells will inhibit plasminogen activation by tissue-type plasminogen activator. Inhibitory activity in the conditioned medium increases with time, and depends upon protein synthesis. Also, the conditioned medium directly inhibits the amidolytic activity of tissue-type plasminogen activator. Inhibitory activity can be removed from conditioned medium by absorption with immobilized tissue-type plasminogen activator.