Shear stress regulates endothelial nitric oxide and superoxide (O₂^−·) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 μg/mL of native LDL were exposed to (1) pulsatile flow with a mean shear stress (τ_ave) of 25 dyne/cm² and (2) oscillating flow at τ_ave of 0. After 4 hours, aliquots of culture medium were collected for high-performance liquid chromatography analyses of electronegative LDL species, described as LDL⁻ and LDL²⁻. In response to oscillatory shear stress, gp91^phox mRNA expression was upregulated by 2.9±0.3-fold, and its homologue, Nox4, by 3.9±0.9-fold (P<0.05, n=4), with a corresponding increase in O₂^−· production rate. The proportion of LDL⁻ and LDL²⁻ relative to static conditions increased by 67±17% and 30±7%, respectively, with the concomitant upregulation of monocyte chemoattractant protein-1 expression and increase in monocyte/BAEC binding (P<0.05, n=5). In contrast, pulsatile flow downregulated both gp91^phox and Nox4 mRNA expression (by 1.8±0.2-fold and 3.0±0.12-fold, respectively), with an accompanying reduction in O₂^−· production, reduction in the extent of LDL modification (51±12% for LDL⁻ and 30±7% for LDL²⁻), and monocyte/BAEC binding. The flow-dependent LDL oxidation is determined in part by the NADPH oxidase activity. The formation of modified LDL via O₂^−· production may also affect the regulation of monocyte chemoattractant protein-1 expression and monocyte/BAEC binding.