—The vascular endothelium is exposed to a spectrum of fluid mechanical forces generated by blood flow; some of these, such as fluid shear stress, can directly modulate endothelial gene expression. Previous work by others and in our laboratory, using an in vitro uniform laminar shear stress model, has identified various shear stress response elements (SSREs) within the promoters of certain endothelial genes that regulate their expression by interacting with various transcription factors, including nuclear factor-κB (NF-κB), early growth response-1 (Egr-1), and activator protein-1 (AP-1, composed of c-Jun/c-Jun and c-Jun/c-Fos protein dimers). In the current study, we have examined the topographical patterns of NF-κB, Egr-1, c-Jun, and c-Fos activation in a specially designed in vitro disturbed laminar shear stress model, which incorporates regions of significant spatial shear stress gradients similar to those found in atherosclerosis-prone arterial geometries in vivo (eg, arterial bifurcations, curvatures, ostial openings). Using newly developed quantitative image analysis techniques, we demonstrate that endothelial cells subjected to disturbed laminar shear stress exhibit increased levels of nuclear localized NF-κB, Egr-1, c-Jun, and c-Fos, compared with cells exposed to uniform laminar shear stress or maintained under static conditions. In addition, individual cells display a heterogeneity in responsiveness to disturbed flow, as measured by the amount of NF-κB, Egr-1, c-Jun, and c-Fos in their nuclei. This differential regulation of transcription factor expression by disturbed versus uniform laminar shear stress indicates that regional differences in blood flow patterns in vivo–in particular, the occurrence of spatial shear stress gradients–may represent important local modulators of endothelial gene expression at anatomic sites predisposed for atherosclerotic development.