Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser¹¹⁷⁹ in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser¹¹⁶, Thr⁴⁹⁷, and Ser⁶³⁵. Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser⁶³⁵, which was consistently slower than that at Ser¹¹⁷⁹. Thr⁴⁹⁷ was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser¹¹⁶. Whereas shear-stimulated Ser⁶³⁵ phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser⁶³⁵ may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.