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 wall shear stress on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled wall shear stress to the surface of endothelial monolayers over a period of 24 hr. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable to S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of approximately 5 dyn/cm², while the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm² shear stress coincided with elevated phosphorylation of Akt and MMP-2 activation. Further, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the PI3K/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced EC invasion into collagen matrices through enhanced Akt and MMP-2 activation.