The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases),which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site-directed chemical cleavage, the SDS-resistant parts of the epitope were located in two fragments of the C-terminal lobe ofNTPDase3 (i.e. Leu220-Cys347 and Cys347-Pro485), which are both required for antibody binding. Additional site-directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399-Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C-terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60-90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.