Evidence is accumulating that extracellular nucleotides act as autocrine/paracrine agents in most tissues, including the kidneys. Several families of surface-located enzymes, collectively known as ectonucleotidases, can degrade nucleotides. Using immunohistochemistry, we have examined the segmental distribution of five ectonucleotidases along the rat nephron. Perfusion-fixed kidneys were obtained from anesthetized male Sprague-Dawley rats. Cryostat sections of cortical and medullary regions were incubated with antibodies specific to the following enzymes: ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1, NTPDase2, NTPDase3, ectonucleotide pyrophosphatase phosphodiesterase 3 (NPP3), and ecto-5'-nucleotidase. Sections were then costained with Phaseolus vulgaris erythroagglutinin (for identification of proximal tubules) and antibodies against Tamm-Horsfall protein (for identification of thick ascending limb), calbindin-D(28k) (for identification of distal tubule), and aquaporin-2 (for identification of collecting duct). The tyramide signal amplification method was used when the ectonucleotidase and marker antibody were raised in the same species. The glomerulus expressed NTPDase1 and NPP3. The proximal tubule showed prominent expression of NPP3 and ecto-5'-nucleotidase in most, but not all, segments. NTPDase2 and NTPDase3, but not NPP3 or ecto-5'-nucleotidase, were expressed in the thick ascending limb and distal tubule. NTPDase3, with some low-level expression of ecto-5'-nucleotidase, was also found in cortical and outer medullary collecting ducts. Inner medullary collecting ducts displayed low-level staining for NTPDase1, NTPDase2, NTPDase3, and ecto-5'-nucleotidase. We conclude that these ectonucleotidases are differentially expressed along the nephron and may play a key role in activation of purinoceptors by nucleotides and nucleosides.