Primary cultures of human hepatocytes and the human hepatocellular cell line Hep G2 are shown to produce fast-acting inhibitors of tissue-type plasminogen activator (tPA) and urokinase. The tPA inhibitory activities in conditioned medium of these liver cell types are very similar to those present in human endothelial cell conditioned medium. They are stable at pH 2.5, have similar dissociation constants with tPA (1.5 to 5 pmol/L), and are similar in thermostability. Addition of tPA to conditioned medium of Hep G2 and endothelial cells that has been depleted of tPA and urokinase reveals a 100 kilodalton tPA-inhibitor complex. The fast-acting tPA inhibitory activity in human plasma has comparable properties, and may originate from the liver or the vascular endothelium or both. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of conditioned medium from hepatocytes, Hep G2, and endothelial cells, additional fibrinolytic inhibition at 52 kilodaltons was visualized. This was not found with human plasma.