In type 2 diabetes, the impairment of vascular repair processes and angiogenesis are due to endothelial progenitor cell (EPC) dysfunction. In this study, we established a quantitative methodology to assess EPC function by using an in vitro 5-(6)-carboxyfluorescein diacetate succinimidyl ester-labeling vessel formation assay. The EPCs were cultured in three different glucose concentrations (100, 189.5, and 295.5 mg/dl of d-glucose) representing normal control and diabetes with either good or poor glycemic control, respectively. We found that the in vitro vessel-forming capacity was impaired in EPCs cultured in high glucose concentrations compared to normal control (43.4 ± 0.8% and 34.7 ± 0.7% vs. 50.8 ± 2.1%). We further studied expression of various genes involved in vessel formation. There was a lower level of angiopoietin 1 gene expression in EPCs cultured in high glucose concentrations. The addition of recombinant angiopoietin 1 significantly increased the vessel-forming capacity of EPCs cultured in high glucose concentration (35.3 ± 2.0% to 48.8 ± 2.7%), whereas the addition of angiopoietin 2 (a competitive inhibitor of angiopoietin 1) impaired the vessel-forming capacity of EPCs cultured in normal glucose concentration (51.8 ± 1.3% to 41.3 ± 0.6%). We conclude that the in vitro vessel-forming capacity of EPCs cultured in high glucose concentration is impaired due to low levels of angiopoietin 1. © 2011 Springer-Verlag.