Pulsations in arterial blood flow expose the endothelium to diverse mechanical forces that may differentially regulate endothelial cell (EC) phenotype. We postulated that pulsatile non-reversing shear stress (typical of the common carotid artery), would produce a more "athero-protective" gene expression pattern compared with steady shear stress of the same mean value. Transcriptional analysis of human umbilical vein endothelial cells (HUVEC) subjected to 24 h of pulsatile shear stress (average = 13 dyne/cm(2), range = 7-25 dyne/cm(2); 1 Hz) or steady shear stress (13 dyne/cm(2)) identified approximately 200 differentially expressed genes. Hierarchical cluster analysis indicated that HUVEC respond similarly to both types of shear stress (Pearson correlation coefficient = 0.785). However, categorization of the differentially expressed genes with Ingenuity Pathways Analysis and with Expression Analysis Systematic Explorer revealed possible differences in nitric oxide (NO) production and signaling. Consistent with gene expression analysis, pulsatile shear stress significantly attenuated NO production relative to steady shear stress (0.77 +/- 0.08, p < 0.01) in HUVEC without significantly altering the levels of intracellular reactive oxygen species (0.95 +/- 0.14, p = 0.65). These results demonstrate that the common carotid flow waveform elicits subtle changes in HUVEC responses to arterial levels of shear stress, which lead to differences in NO production.