Adenosine coordinates organ metabolism and blood supply, and it modulates immune responses. In the kidney it mediates the vascular response elicited by changes in NaCl concentration in the macula densa region of the nephron, thereby serving as an important regulator of GFR. To determine whether adenosine formation depends on extracellular nucleotide hydrolysis, we studied NaCl-dependent GFR regulation (tubuloglomerular feedback) in mice with targeted deletion of ecto-5′-nucleotidase/CD73 (e-5′NT/CD73), the enzyme responsible for adenosine formation from AMP. e-5′NT/CD73^–/– mice were viable and showed no gross anatomical abnormalities. Blood pressure, blood and urine chemistry, and renal blood flow were not different between e-5′NT/CD73^+/+ and e-5′NT/CD73^–/– mice. e-5′NT/CD73^–/– mice had a significantly reduced fall in stop flow pressure and superficial nephron glomerular filtration rate in response to a saturating increase of tubular perfusion flow. Furthermore, whereas tubuloglomerular feedback responses did not change significantly during prolonged loop of Henle perfusion in e-5′NT/CD73^+/+ mice, a complete disappearance of the residual feedback response was noted in e-5′NT/CD73^–/– mice over 10 minutes of perfusion. The contractile response of isolated afferent arterioles to adenosine was normal in e-5′NT/CD73^–/– mice. We conclude that the generation of adenosine at the glomerular pole depends to a major extent on e-5′NT/CD73–mediated dephosphorylation of 5′-AMP, presumably generated from released ATP.