Megjegyzés: Abstract Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides. Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP) to adenosine in primary somatosensory neurons. Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons. Ectonucleoside triphosphate diphosphohydrolases (ENTPDs) comprise a class of enzymes that dephosphorylate extracellular ATP and ADP. Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3) was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG), in the dorsal horn of the spinal cord, and in free nerve endings in the skin. To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse. This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder. However, DRG and spinal cord tissues from Entpd3-/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates. Additionally, using fast-scan cyclic voltammetry (FSCV), adenosine production was not impaired in the dorsal spinal cord of Entpd3-/- mice when the substrate ADP was applied. Further, Entpd3- /- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3-/- mice showed a modest reduction in β-alanine-mediated itch. Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord. Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.