The apparent tendency of atherosclerotic lesions to form in complex blood flow environments has led to many theories regarding the importance of hemodynamic forces in endothelium-mediated atherosusceptibility. The effects of shear stress magnitude and spatial shear stress gradient on endothelial cell gene expression in vitro were examined in this study. Converging-width flow channels were designed to impose physiological ranges of shear stress gradient and magnitude on porcine aortic endothelial cells, and real-time quantitative PCR was performed to evaluate their expression of five genes of interest. While vascular cell adhesion molecule-1 (VCAM-1) expression was insensitive to either variable, each of the remaining genes exhibited a unique dependence on shear stress magnitude and gradient. Endothelial nitric oxide synthase (eNOS) showed a strong positive dependence on magnitude, but was insensitive to gradient. The expression of c-jun was weakly correlated with magnitude and gradient, without an interaction effect. Monocyte chemoattractant protein-1 (MCP-1) expression varied inversely with gradient and also depended on the interaction of gradient with magnitude. Intercellular adhesion molecule-1 (ICAM-1) expression also exhibited an interaction effect, and increased with shear magnitude. These results support the notion that vascular endothelial cells are able to sense shear gradient and magnitude independently.