The effect of laminar flow on the regulation of von Willebrand Factor (vWF) of cultured human umbilical vein endothelial cells (HUVECs) was studied. Confluent endothelial monolayers were exposed to shear stresses (0.2 and 1.0 Pa) from 2 to 24 h. VWF was labelled with indirect immunofluorescence method and observed with 3D fluorescence microscopy. The distribution of VWF and the cytoskeleton organization were observed simultaneously by double fluorescence labelling. More actin stress fibers and an increased release of VWF appeared in the cells exposed to how at the same time. The qualitative and quantitative results showed that there was not only a shear-dependent regulation but also a time-dependent modification. For a short-time shear stimulation, both 0.2 Pa and 1.0 Pa shear stresses induced a release of VWF from the endothelial cells. In contrast, after 24 h exposure to 1.0 Pa shear dow, vWFs were much more in the cells than that in the cells exposed to 0.2 Pa for 24 h (p < 0.01) or that in the control cells (p < 0.05). TNF-alpha caused a decrease of VWF and Weibel-Palade bodies in the cells.