Endothelium constitutes a highly specialized organ that lines the vascular system and lymphatic channels. This organ is a complex network of arteries, veins, and microvessels that differ in size, structure, and function. The unique and strategic location imposes functional demands on the endothelium that are far greater than just being a passive barrier. Endothelial cells have the ability to differentiate both in structure and function in response to the needs of diverse tissue environments, making this organ extremely heterogeneous. Although vascular endothelial cells share certain common properties, they differ in regard to structure, antigenic and cell surface determinants, adhesion molecules, and metabolic function. The unique cell surface profiles expressed by endothelial cells in different tissue locations can be recognized by specific populations of circulating leukocytes or tumor cells, which contribute to their arrest and invasion patterns. This article attempts to review our current understanding of endothelial cell heterogeneity and its significance to patterns of leukocyte and tumor cell trafficking.