Background: S100A1, a Ca 2+ -sensor protein in endothelial cell, regulates endothelial function. S100A1 knockout mice (KO) are hypertensive with impaired nitric oxide (NO) production and endothelial-dependent vasodilatation. We examine the impact of S100A1 on phosphorylation of Ser 1177 and Thr 495 , positive and negative regulatory residues, respectively, in endothelial nitric oxide synthase (eNOS) and on expression of diverse genes involved in eNOS signaling. Methods and Results: Endothelial cells (EC) were isolated and from pulmonary vasculature of wild-type (WT) and KO mice. WT EC exhibited higher proliferative rates than KO measured by WST-method, and expressed as absorbance/5000 cells/well (0.8923±0.091 vs. 0.268±0.055, p<0.05, n=3) and higher NO production (2214.00±1016.17 vs. 566.75±243.79 nM, p<0.05, n=6). Lower eNOS activity in KO was associated with higher and lower basal phosphorylation at Thr 495 and Ser 1177 , respectively. Bradykinin (100 nM) resulted in rapid (1 minute) 3-fold higher maximal phosphorylation of Ser 1177 in WT vs. KO with equivalent rapid dephosphorylation of Thr 495 in both cell types. By contrast, acetylcholine (10 μ M) at 30 seconds produced more robust dephosphorylation of Thr 495 in WT compared to KO, with comparable effects on Ser 1177 phosphorylation. In KO versus WT EC, PCR microarray analysis demonstrated greater than 2-fold increase in mRNA expression of caveolin-1, NADPH oxidase 1, glutathione peroxidase, and calmodulin-1. KO EC exhibited a 5-fold reduction in eNOS mRNA, confirmed by a reduction in eNOS protein (4.02±0.19 fold, n=3). Conclusion: Absence of S100A1 in endothelial cells results in impaired proliferation, decreased NO availability, and regulation of gene expression that contributes or, alternately, compensates for this phenotype. Lack of S100A1 also impairs co-ordinated eNOS phosphorylation at baseline, and in response to agonists, that augments NO production. Thus, S100A1 plays a multifactorial and novel role in controlling NO synthesis and the endothelial phenotype.