Introduction: The hemodynamic environment of blood vessels is a crucial feature of endothelium-mediated vasoregulation. Flow-associated laminar shear stress (LSS) imparts an atheroprotective stimulus on the vascular endothelium, causing realignment of endothelial cells in the direction of flow and strengthening of intercellular tight junction barriers. Conversely, disturbed shear stress (DSS), a contributing factor to vascular diseases such as atherosclerosis and coronary artery disease, prevents cellular realignment and causes barrier weakening. Equally crucial to endothelium-mediated vasoregulation are vasoactive peptidases. Evidence is emerging to show that peptidase expression and activity within the vasculature is regulated in a highly coordinated manner by vessel hemodynamic forces (Fitzpatrick, et al. 2009). Indeed, this phenomenon likely has significant consequences for the net balance of pressor versus depressor peptides within the endothelium, subsequently leading to downstream effects on vessel wall tone and remodeling events. Of particular importance are the „thermolysin-like‟ family of zinc metallopeptidases belonging to the Clan MA (Barrett, Rawlings and O'Brien 2001), which hydrolyse peptide hormone substrates of less than 40 amino acids, and which are known to contain a single copy shear stress response element (-GAGACC-) within their promoter regions. In the current study it is therefore hypothesised that, in view of their opposing physiological actions in vivo, shear stress can modulate vascular endothelial expression of neprilysin (NEP, EC3.4.24.11) and thimet oligopeptidase (TOP, EC3.4.24.15) in an inverse manner, with functional consequences for endothelial homeostasis. Results: Exposure of cultured bovine/human aortic endothelial cells (BAECs/HAECs) to LSS of varying magnitude and duration (0-10 dynes/cm2, 0-48 hrs) resulted in dose- and time-dependent modulation of NEP (down-regulation) and TOP (up-regulation) expression as monitored via qPCR, immunocytochemistry, and Western blotting. Using a luciferase reporter vector (pGL3-TOP, full-length promoter -901/+120), LSS was also found to substantially increase TOP promoter activity. By contrast, DSS had opposite effects to LSS on the expression of NEP (up-regulation) and TOP (down-regulation). We subsequently employed pharmacological antioxidants (N-acetyl-L-cysteine), inhibitors (Rac1/NSC23766, NADPH oxidase/apocynin) and siRNAs (p47Phox) in shearing experiments to confirm a pivotal role for NADPH oxidase-mediated superoxide production in the LSS mechanotransduction of these reciprocal events. Finally, using a TOP siRNA, an investigation of how LSS-induced TOP up-regulation impacts on endothelial functions (i.e. proliferation and bradykinin hydrolysis) was carried out, and points to functional consequence between these events under shear. Conclusions: This study indicates that the expression of NEP and TOP, zinc metallopeptidases of high physiological relevance to the endothelium, are regulated in an opposing manner by shear stress, with potential consequences for endothelial function and flow-dependent vascular pathologies.