Extracellular nucleotides regulate diverse biological functions and are important in the regulation of liver metabolism, hepatic blood flow, and bile secretion. Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) hydrolyze extracellular nucleotides and are therefore potential regulators of nucleotide-mediated signaling. To examine this, we have contrasted the structural and functional distributions of the 2 characterized membrane-bound NTPDases NTPDase1 and NTPDase2 within the rat liver. Hepatic expression of NTPDase2 was determined and contrasted to NTPDase1 using confocal immunofluorescence, immunoelectron microscopy, reverse-transcription polymerase chain reaction, Northern blot analysis, Western blot analysis, and functional assays. NTPDase2 was expressed in the periportal region surrounding intrahepatic bile ducts, whereas NTPDase1 was found in hepatic arteries, portal veins, and hepatic central veins, consistent with its known vascular distribution. Functional and molecular expression of NTPDase2 was shown in portal fibroblasts near basolateral membranes of bile duct epithelia. In conclusion, NTPDase2 is expressed in a novel cellular compartment surrounding intrahepatic bile ducts, namely portal fibroblasts. This distribution may represent a previously unrecognized mechanism for regulation of nucleotide signaling in bile ducts and other epithelia.