Aim To investigate the isolation and culture of endothelial progenitor cells (EPC) from peripheral blood in patients with coronary heart diseases (CHD), and to determine cell shape, amount, activity and clusters. Methods Mononuclear cells were isolated from peripheral blood of patients with CHD by Ficoll-density contrifugation. The isolated cells were cultured in 1640 medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The EPC specific surface mark CD34 and KDR were assessed by fluorescence activated cell sorter (FACS) analysis. EPC were characterized as adherent cells double positive for Dil marked acetylated low density lipoprotein (acLDL-Dil) uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPC proliferation were assayed by MTT assay. Results The number of EPC was significantly reduced in patients with CHD compared with control subjects (23.1±1.8 vs 56.7±2.4, P<0.05), and the number of cell clusters was also reduced in patients with CHD compared with control subjects (14.7±2.5 vs 24.2±1.7, P<0.05). The proliferation was also impaired in patients with CHD. Conclusion EPC were cultured and differentiated at specific conditions derived from mononuclear cells of peripheral blood.