Although hydrogen peroxide (H₂O₂) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding -galactosidase (Adgal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by -galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adgal-transduced cells (536.8 ± 31 vs. 1,875.1 ± 132.9 vs. 1,347.5 ± 93.7 dpm/well, respectively; P < 0.05 vs. control and Adgal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 ± 1.8% of cell counts in control, P < 0.05; and 45 ± 2% of cell count in Adgal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H₂O₂ (50 µM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H₂O₂ is an important modulator of endothelial cell proliferation.