Flow impingement at arterial bifurcations causes high frictional force (or wall shear stress, WSS), and flow acceleration and deceleration in the branches create positive and negative streamwise gradients in WSS (WSSG), respectively. Intracranial aneurysms tend to form in regions with high WSS and positive WSSG. However, little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile gene expression in cultured ECs exposed to positive or negative WSSG for 24 hours in a flow chamber where WSS varied between 3.5 and 28.4 Pa. Gene ontology and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of pro-inflammatory genes. To determine if similar responses occur in vivo, we examined EC proliferation and expression of the matrix metalloproteinase, ADAMTS1, under high WSS and WSSG created at the basilar terminus of rabbits after bilateral carotid ligation. Precise hemodynamic conditions were determined by computational fluid dynamic simulations from 3D angiography and mapped on immunofluorescence staining for the proliferation marker, Ki-67, and ADAMTS1. Both proliferation and ADAMTS1 were significantly higher in ECs under positive WSSG than in adjacent regions of negative WSSG. Our results indicate that WSSG elicits distinct EC gene expression profiles and particular biological pathways including increased cell proliferation and matrix processing. Such ECs responses may be important in understanding the mechanisms of intracranial aneurysm initiation at regions of high WSS and positive WSSG.