Chronic exposure of the skeletal muscle microcirculation to elevated shear stress-induces angiogenesis. Previous studies observed that shear stress-induced capillary growth involves luminal sprouting, or internal division, of the capillaries, which is characterized by a minimal proliferative response and the retention of an intact basement membrane. Matrix metalloproteinases (MMPs) are associated with the process of abluminal sprouting angiogenesis, but may not be required for the process of luminal division during capillary growth. We analyzed the production of MMP-2, using both the in vivo model of prazosin-induced angiogenesis in rat skeletal muscle, and cultured microvascular endothelial cells exposed to laminar shear stress. We found that MMP-2 was not elevated in capillaries of shear stress-stimulated skeletal muscle, despite a significant increase in capillary number in response to a shear stress stimulus. In cultured microvascular endothelial cells, MMP-2 mRNA and protein levels were attenuated significantly in response to shear stress exposure. This effect on MMP-2 was reversed by nitric oxide (NO) synthase inhibition using LNNA. In contrast, exposure of static cultures of endothelial cells to NO donors significantly reduced MMP-2 production. Shear stress exposure and NO donors both modified phosphorylation levels of several members of the MAPK family. Treatment of shear stress-exposed cells with the p38 MAPK inhibitor, SB203580, abolished the shear stress-mediated reduction in MMP-2 mRNA. Thus, our data provide strong evidence that elevated shear stress inhibits MMP-2 production in microvascular endothelial cells, an effect that is mediated by signal pathways involving both production of NO and activation of p38 MAPK. J. Cell. Physiol. © 2006 Wiley-Liss, Inc.