The aim of our study was to evaluate the relevance of tissue- and species-specific endothelial cells (EC) in order to study EC-dependent mechanisms in inflammatory-mediated tissue injury. We established an isolation protocol for highly purified EC (pEC) preparations of different origin, and compared EC-specific inflammatory responses. Fluorescence-activated cell separation was used to obtain pEC cultures from different human arterial (coronary artery, internal thoracic artery) and venous (umbilical vein, saphenous vein) vessels. All pEC were analyzed for growth kinetics, morphology, release of cytokines/chemokines and expression of E-selectin. For all different EC cultures purities of 99% were reproducibly achieved. The EC isolation did not affect EC growth, morphology, and function. However, characterization of pEC from different vessel materials revealed an intrinsic, tissue-specific functional heterogeneity of EC cultures. Despite an arterial and venous difference in the secretion of IL-8 and MCP-1, especially EC from coronary arteries produced significantly more IL-6 as compared to other EC types, independent of age, gender and disease of the cell donors. In contrast, the expression of E-selectin was not affected. We conclude that the proposed isolation protocol allows the generation of a pEC bank enabling us to study tissue-specific aspects at the level of the endothelium.