The objective of the study was to determine the role of the angiopoietins in the regulation of gelatinase expression during angiogenesis, and whether inhibition of the angiopoietin/Tek interaction in vivo can suppress the extent of retinal neovascularization. Retinal microvascular endothelial cells were treated with angiopoietins and examined for the production of gelatinases. The effects of inhibiting angiopoietin binding to the Tie-2 receptor was studied in newborn mice with experimentally induced retinal neovascularization. Animals were treated with an ip injection of the Tie-2 antagonist, muTek delta Fc, while oxygen-exposed mice treated with similar concentrations of murine IgG were used as controls. The effect of muTek delta Fc on the gelatinase expression in the retina was examined by real-time RT-PCR analysis. The stimulation of cultured retinal endothelial cells with Ang-1 and -2 resulted in the increased expression of matrix metalloproteinase (MMP)-9. Ang-2 expression was up-regulated in experimental animals during the period of angiogenesis and was the greatest on Day 17 (the time of maximal angiogenic response). Histologic analysis of mice treated with the Tie-2 antagonist, muTek delta Fc, showed significant (87%; p = 0.001) inhibition of retinal neovascularization, and the response was dose-dependent. In vitro binding data support the fact that both Ang-1 and Ang-2 bind with high avidity to muTek delta Fc. The RT-PCR analysis of the retinas of the Tek-treated animals showed a similar (80%; p = 0.001) inhibition of the MMP-9 expression, which correlated with the decrease in angiogenesis. The up-regulation of gelatinases in microvascular endothelial cells by Ang-2 may be an important early response during the development of retinal neovascularization. Inhibition of the binding activity of the angiopoietins in vivo suppressed retinal neovascularization concomitant with a reduction in the expression of MMP-9.