Previous investigations have compared the levels of endothelial nitric oxide synthase (eNOS) protein among different branch orders of the coronary arterial tree. In these studies, eNOS protein levels were determined in whole vessels and thus were likely influenced by variable amounts of non‐endothelial cell types in the lysate as vessel wall thickness varied. For the current study, we isolated endothelial cells from both coronary conduit arteries and microvasculature to test the hypothesis that the content of eNOS protein remains similar in coronary endothelial cells regardless of vessel radius. Porcine hearts were obtained from a local abattoir and transported to the lab in iced Krebs solution. Large (conduit) and small arteries as well as arterioles were dissected free of myocardium and homogenized as whole vessels. Additionally, endothelial cells were isolated from both conduit arteries and left ventricular myocardium by mincing the tissue into small pieces, digesting with collagenase, followed by endothelial cell isolation using biotinylated‐anti‐CD31 and streptavidin‐coated paramagnetic beads. Purity of isolated coronary endothelial cells was confirmed by immunofluorescence and immunoblot. In whole vessel lysate, immunoblot analysis revealed that protein content for eNOS was significantly greater in arterioles compared to small arteries and large arteries. In contrast, eNOS protein levels in endothelial cells isolated from coronary microvasculature were not significantly different from those isolated from large conduit arteries. Taken together, these data indicate that endothelial cell eNOS protein content is comparable regardless of whether endothelial cells are isolated from the coronary microcirculation or conduit arteries. Furthermore, use of whole vessel lysate among different branch orders to compare levels of endothelium‐specific proteins may be unsuitable due to variable contamination by other cell types.Support or Funding InformationNIH R01‐HL064931