Antiangiogenic drugs have been used successfully for the treatment of colorectal cancer (CRC) and several other tumor types. Until recently, viable tumor endothelial cells (TEC) and normal endothelial cells of uninvolved colon tissue (NEC) from the same patient have not been available to optimize treatment strategies in vitro. Here, we describe a protocol for the isolation of TEC and NEC. These cells were isolated at a very high purity via magnetic cell sorting of tissue samples obtained from surgical specimens of patients suffering from CRC. Isolated TEC and NEC expressed CD31, CD105, VE-cadherin, VCAM-1, ICAM-1, and E-selectin, formed capillaries in basal membrane extract, and were able to take up acetylated LDL. They were negative for podoplanin, CD45, CD68, and CK-20, indicating blood vessel endothelial lineage. Expression of vWF was more pronounced in NEC cultures, whereas vWF was absent or only slightly expressed in all TEC cultures in vitro. Lower intracellular concentrations of vWF were also detected in TEC as compared to NEC at the tissue level. The latter finding demonstrated that differential features of TEC and NEC in vivo are stably perpetuated in culture. The isolated endothelial cell cultures may provide a useful in vitro model system to elucidate epigenetic effects on angiogenesis in cancer and to optimize antiangiogenic therapy.