Previously, we have demonstrated that increased superoxide generation plays a role in the nitric oxide (NO)-mediated inhibition of endothelial NO synthase (eNOS) in endothelial cells (ECs) and that the overexpression of SOD1 could reduce the inhibitory effect of NO. However, SOD1 overexpression did not completely abolish the inhibition of eNOS by NO, indicating the presence of other inhibitory mechanisms. Because superoxide can be dismutated into hydrogen peroxide (H2O2), in this study we determined whether exposure of ECs to NO resulted in increased generation of H2O2 and the potential role of H2O2 in eNOS inhibition. Our results indicated that H2O2 levels were increased in response to NO. Using adenoviral-mediated infection, we demonstrated that catalase overexpression both increased basal eNOS activity in the absence of NO and provided a significant protective effect on eNOS activity in the presence of NO. This protective effect was associated with a significant decrease in H2O2 levels in the presence of NO. In conclusion, our results indicate that increased levels of H2O2 may be involved in the inhibition of eNOS by NO and that the scavenging of H2O2 may be useful to prevent eNOS inhibition during treatments that involve inhaled NO or NO donors.