Endothelial cells, once stimulated with thrombin, are resistant to subsequent stimulation. After a recovery period of about 60 min the cells are sensitized again for activation by thrombin. The resensitization is independent of the receptor de novo synthesis. Therefore an intracellular pool of thrombin receptors that is possibly co-localized with the Golgi apparatus has been assumed. Brefeldin A (BFA) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organization of the Golgi apparatus. Accordingly we have examined the effects of BFA on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. The von Willebrand factor (VWF) release from Weibel-Palade vesicles and the intracellular calcium mobilization were used as physiological parameters of thrombin receptor activation. The addition of BFA (2 μg/ml) to endothelial cells or the reduction of the incubation temperature from 37°C to 16°C blocked the receptor response regeneration almost completely.