Endothelial cells are subjected to biochemical and mechanical stimuli which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid shear wall stress (WSS) on the invasion of human umbilical vein endothelial cells (HUVECs) into three-dimensional collagen matrices. A collagen gel was incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of HUVEC monolayers over a period of 24hr. Cell invasion into the gels required the presence of S1P, with the effects of S1P being enhanced by WSS to an extent comparable to growth factor (VEGF + FGF-2) stimulation. The number of invading cells depended on the magnitude of WSS, with a maximal induction at a WSS of 5 dyne/cm2. The enhancement of invasion by WSS coincided with the phosphorylation of Akt and MMP-2 activation. These results provide evidence that WSS is a positive modulator of S1P-induced EC invasion into collagen gels and may contribute to the localization of sprouting angiogenesis at regions of low WSS such as post-capillary venules.