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 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. Ang-2 mRNA is 1.6-fold upregulated by low shear stress (1 dyn/cm², 24 h, n=10), but downregulated to 57±10% by high shear stress (max. 30 dyn/cm², 24 h, n=16). 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. Downregulation of Ang-2 by high shear stress involves PKC. Furthermore, dose-dependent stimulation with VEGF induced Ang-2 mRNA. The upregulation of Ang-2 by VEGF was inhibited by application of high shear stress (30 dyn/cm², 24 h) (con: 100±20%, VEGF: 421±41%*, VEGF+shear stress: 227±50%^#, *P<0.05 vs. con, ^#P<0.05 vs. VEGF, n=5). Low levels of shear stress causes upregulation, but high shear stress downregulation of Ang-2 in HUVEC. Our data suggest a role of PKC and VEGF in shear stress-dependent regulation of Ang-2. These data support a dose-dependent regulation of genes involved in the differentiation of endothelial cells by shear stress.