Studies in mice with null mutations of adenosine 1 receptor (A1AR) or ecto-5’-nucleotidase genes suggest a critical role of adenosine and its precursor 5’-AMP in tubulo-vascular signaling. To assess whether the source of juxtaglomerular nucleotides can be traced back to ATP dephosphorylation, experiments were performed in mice with a deficiency in NTPDase1/CD39, an ecto-ATPase catalyzing the formation of AMP from ATP and ADP. Urine osmolarity and GFR were indistinguishable between NTPDase1/CD39-/- and wild type (WT) mice. Maximum tubuloglomerular feedback (TGF) responses, as determined by proximal tubular stop flow pressure (PSF) measurements, were reduced in NTPDase1/CD39 -/- mice compared to controls (10.5 ± 1.2 vs. 4.2 ± 0.9 mm Hg, respectively; p=.0002). Residual TGF responses gradually diminished after repeated changes in tubular perfusion flow averaging 2.9 ± 0.9 (on-response) and 3.5 ± 1.1 mm Hg (off-response) after the second, and 2.2 ± 0.5 (on-response) and 1.5 ±0.8 mm Hg (off-response) after the third challenge, whereas no fading of TGF responsiveness was observed in WT mice. Macula densa-dependent and pressure-dependent inhibition of renin secretion, as assessed by acute salt loading and phenylephrine injection, respectively, were intact in NTPDase1/CD39-deficient mice. In summary, NTPDase1/CD39-deficient mice showed a markedly compromised TGF regulation of GFR. These data support the concept of an extracellular dephosphorylation cascade during tubular-vascular signal transmission in the JGA which is initiated by a regulated release of ATP from MD cells and results in adenosine-mediated afferent arteriole constriction.