Local differences in shear stress can modulate the endothelial phenotype. Angiopoietin-2 (Ang-2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. However, the regulation of Ang-2, especially in response to shear stress, is not well understood. Therefore, we analyzed the regulation of Ang-2 by shear stress. Human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress (1 to 30 dyn/cm2 for up to 24 h) in a cone-and-plate viscometer. The mRNA expression of Ang-2 and FOX01 was determined by RT-PCR, Ang-2 and FOXO1 protein expression by Western blot, and Ang- 2 peptide release by ELISA. The role of protein kinase B (AKT) was analyzed using PI3K/AKT inhibitor LY 294002. FOXO1 localization was further analysed by immunofluorescence. Ang-2 mRNA is 1.6-fold upregulated by low shear stress (1 dyn/cm2, 24 h), but downregulated to 57±10% by high shear stress (max. 30 dyn/cm2, 24 h). Ang-2 protein expression and release is induced by long-term (24 h) low shear stress (1 dyn/cm2), but downregulated by high laminar shear stress (30 dyn/cm2) as well. FOXO1 mRNA and protein expression is not regulated by shear stress, but FOXO1 protein translocates out of the nucleus after shear stress (30 dyn/cm2, 24 h). Our data support a role of FOXO1 translocation in shear stress- dependent regulation of Ang-2.