Nitric oxide (NO) is a highly reactive molecule involved in a variety of physiological and pathophysiological processes, such as inflammation. eNOS-produced NO has anti-inflammatory properties and play a role in vascular homeostasis. Through a variety of mechanisms, tumor necrosis factor alpha (TNF-α) has been shown to induce inflammation in HUVECs. The purpose of this study was to investigate the role of NO in HUVEC cells under inflammatory conditions induced by TNF-α. To identify TNF-α induction mechanisms, the phosphorylation of ERK, Akt, and eNOS was investigated. Sildenafil and L-NAME used to examine the role of NO in this process. In TNF-α- induced HUVEC cells, cell viability, nitrite/nitrate production, phosphorylated and total ERK, Akt, and eNOS levels were measured with or without sildenafil and L-NAME. At 20 and 40 ng/ml concentrations, TNF-α increased nitrite/nitrate and decreased cell viability (p<0.05). Sildenafil and L-NAME had no effect on cell viability and they both decreased nitrite/ nitrate in HUVEC that had been stimulated by TNF-α. TNF-α had no effect on the phosphorylation of ERK, Akt, and eNOS, but it did increase total eNOS levels. The phosphorylation of ERK, Akt, and eNOS was unaffected by sildenafil and L-NAME; however, L-NAME decreased total eNOS. According to our findings, there is no known direct relationship between TNF-α, sildenafil, or L-NAME and protein phosphorylation.