The growth of new blood vessels, known as angiogenesis, is a dynamic but highly regulated process involving many different regulatory pathways. Endothelial cell migration and proliferation is also essential for this process to occur. Studying the behavior of endothelial cells and how they repond to the factors involved in angiogenesis is important in understanding how the process is controlled. The ability to generatic primary endothelial cells allows for such detailed studies. In this chapter, we present the method for endothelial cell isolation that we have found to be successful and that we routinely employ to isolate and culture primary endothelial cells.