Background/Aims: Human coronary artery-derived endothelial cells (ECs) seem to be the most appropriate cells for the pathogenesis study of coronary artery disease. But limited availability of endothelial tissue is a major constraint. In this study, we developed a method to isolate human coronary artery ECs in vivo from patients. Methods: Coronary guidewires were used to obtain EC samples from coronary arteries in 76 patients. Cells were eluted from wire tips and purified by immunomagnetic beads. Von Willebrand factor and CD31 were used as immunocytochemical markers to identify cells as endothelium. Cell viability was evaluated in terms of cell membrane integrity, energy-dependent uptake of DiI-labeled acetylated low-density lipoprotein, and apoptosis. Nitric oxide synthase (eNOS) expression and nitric oxide (NO) production of cells were detected to evaluate cell function. Results: About 96 coronary artery ECs were obtained per guidewire. Cells manifested endothelial morphology and immunoreactivity for von Willebrand factor and CD31 with good viability. But eNOS expression and NO production of cells were decreased. Conclusions: Viable coronary endothelium could be obtained during routine percutaneous coronary interventions combined with immunomagnetic beads. These cells may be used for advanced cellular functional analyses such as immunocytochemistry and molecular biology. Such information could aid in understanding mechanisms of coronary artery diseases.