Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides to the respective nucleosides. Within the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and characterized. In this article, we specifically address the cell surface-located members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) family (NTPDase1,2,3, and 8). The molecular identification of individual NTPDase subtypes, genetic engineering, mutational analyses, and the generation of subtype-specific antibodies have resulted in considerable insights into enzyme structure and function. These advances also allow definition of physiological and patho-physiological implications of NTPDases in a considerable variety of tissues. Biological actions of NTPDases are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides and consequent effects on P2-receptor signaling. It further appears that the spatial and temporal expression of NTPDases by various cell types within the vasculature, the nervous tissues and other tissues impacts on several patho-physiological processes. Examples include acute effects on cellular metabolism, adhesion, activation and migration with other protracted impacts upon developmental responses, inclusive of cellular proliferation, differentiation and apoptosis, as seen with atherosclerosis, degenerative neurological diseases and immune rejection of transplanted organs and cells. Future clinical applications are expected to involve the development of new therapeutic strategies for transplantation and various inflammatory cardiovascular, gastrointestinal and neurological diseases.