We previously reported that association of endothelial nitric oxide synthase (eNOS) with actin increases eNOS activity. In the present study, the regulation of activity of eNOS by the actin cytoskeleton during endothelial growth was studied. We found that eNOS activity in pulmonary artery endothelial cells (PAEC) increased when cells grew from preconfluence to confluence. The eNOS activity was also much greater in PAEC in higher density than those in lower density, suggesting that the increase in eNOS activity during cell growth is caused by increase in cell density. Although eNOS protein contents were also increased when endothelial cells grew from preconfluence to confluence, the magnitude of increase in eNOS activity was much higher than the increase in eNOS protein content, suggesting that post-translational mechanisms played an important role in the regulation of eNOS activity during endothelial growth. Confocal fluorescence microscopy revealed that eNOS was colocalized with G-actin in preconfluent cells in the perinuclear region, and with both G-actin in the perinuclear area and with cortical F-actin in the plasma membrane in confluent cells. There was more We previously reported that association of endothelial nitric oxide synthase (eNOS) with actin increases eNOS activity. In the present study, the regulation of activity of eNOS by the actin cytoskeleton during endothelial growth was studied. We found that eNOS activity in pulmonary artery endothelial cells (PAEC) increased when cells grew from preconfluence to confluence. The eNOS activity was also much greater in PAEC in higher density than those in lower density, suggesting that the increase in eNOS activity during cell growth is caused by increase in cell density. Although eNOS protein contents were also increased when endothelial cells grew from preconfluence to confluence, the magnitude of increase in eNOS activity was much higher than the increase in eNOS protein content, suggesting that post-translational mechanisms played an important role in the regulation of eNOS activity during endothelial growth. Confocal fluorescence microscopy revealed that eNOS was colocalized with G-actin in preconfluent cells in the perinuclear region, and with both G-actin in the perinuclear area and with cortical F-actin in the plasma membrane in confluent cells. There was more-actin co-immunoprecipitated with eNOS in the Triton-X-100-soluble fraction in the confluent cells in later growth phase and in high density. Decrease in eNOS association with -actin by silencing -actin expression using -actin siRNA causes inhibition of eNOS activity, NO production, and endothelial monolayer wound repair in PAEC. Moreover, incubation of PAEC with cytochalasin-D and jasplakinolide resulted in increases in eNOS/actin association and in eNOS activity without changes in eNOS protein content. Yeast two-hybrid experiments suggested a strong association between the eNOS oxygenase domain and -actin. These results indicate that the increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC. actin co-immunoprecipitated with eNOS in the Triton-X-100-soluble fraction in the confluent cells in later growth phase and in high density. Decrease in eNOS association with -actin by silencing -actin expression using -actin siRNA causes inhibition of eNOS activity, NO production, and endothelial monolayer wound repair in PAEC. Moreover, incubation of PAEC with cytochalasin-D and jasplakinolide resulted in increases in eNOS/actin association and in eNOS activity without changes in eNOS protein content. Yeast two-hybrid experiments suggested a strong association between the eNOS oxygenase domain and -actin. These results indicate that the increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC.