Atherosclerotic plaques are initiated by pro-inflammatory endothelial activation at arterial regions subjected to non-uniform shear stress. We applied the in vitro flow-through cell culture slides to investigate whether different patterns of shear stress affect the secreted cytokine and chemokine profile in endothelial cells. Human umbilical vein endothelial cells (ECs) were exposed to 24 h of flow in straight or bifurcating flow-through slides, in some experiments followed by 6 h stimulation with 2.5 ng/mL TNF-α. IL-1β, IL-6, IL-8, IL-12p70, MIP-1α, MCP-1 and sICAM-1 were measured in conditioned medium samples by flow cytometry using antibodies conjugated with fluorescent beads. The release of IL-1β, IL-12p70 and MIP-1α from endothelial cells exposed to shear stress was below detectable levels. Strongly increased level of sICAM and significantly increased IL-8 concentration were detected in conditioned medium from endothelial cells exposed to flow in bifurcating slides as compared with cells grown under laminar flow in straight channels. The release of IL-6 and MCP-1 was not significantly induced in bifurcating slides. Treatment with TNF-α for 6 h induced 2-3 fold increase in secreted chemokines and cytokines. In particular, significantly increased MCP-1 and increased IL-8 levels were released from endothelial cells grown in bifurcating slides. This release was partly prevented in cells grown in straight channels, i.e. under exposure to laminar flow only. Although the endothelial monolayer areas exposed to non-uniform shear stress are relatively small, the activation of cells in these regions is strong enough to produce a detectable change in cytokine and chemokine profile, which represents the earliest step in atherogenic endothelial dysfunction.