Antiangiogenic drugs have been used successfully for the treatment of colorectal cancer (CRC). Viable tumor endothelial cells (TEC) and normal endothelial cells (NEC) of uninvolved colon tissue of the same patient have not been available to optimize treatment strategies in vitro. Therefore, our target was to establish a protocol for the isolation of TEC and NEC. These cells were isolated with very high purity via magnetic cell sorting of tissue samples obtained from CRC and healthy colon of eight patients. 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 low-density lipoprotein. They were negative for podoplanin, CD45, CD68 and cytokeratin-20 indicating blood vessel endothelial lineage. Intense staining of von Willebrand factor (vWF) was observed in five of eight NEC cultures, whereas vWF was absent or only slightly expressed in all TEC cultures in vitro. Low intracellular concentration of vWF was also detected in TEC and NEC at the tissue level. This demonstrated that differences exhibited by TEC and NEC in vivo are stably perpetuated in culture. The isolated cultures may provide a useful in vitro model to elucidate epigenetic effects on angiogenesis in cancer and to optimize antiangiogenic therapy.