Tubuloglomerular feedback (TGF) describes a sequence of events linking salt concentrations in tubular fluid at the macula densa to the vascular tone of the afferent arteriole and thus to the glomerular filtration rate (GFR) of the same nephron. The signal transduction pathways of TGF remain incompletely understood, but both ATP release from macula densa cells and local formation of adenosine were suggested to be involved in the process. To test the role of extracellular formation of adenosine by ecto-5'-nucleotidase (cd73) in TGF, in regulation of GFR, and in tubular reabsorption, renal clearance and micropunture experiments were performed in cd73 wild-type (cd73(+/+)) and knockout mice (cd73(-/-)). The cd73(-/-) mice presented normal mean arterial blood pressure, but modestly lower whole kidney and single nephron GFR (SNGFR). Fractional reabsorption of Na(+) and K(+) up to the late proximal tubule, distal tubule, as well as urine were not significantly different between cd73(-/-) and cd73(+/+) mice. Lack of cd73 resulted in a diminished TGF response, as indicated by smaller changes of stop-flow pressure in response to increasing loop of Henle perfusion from 0 to 25 nl/min, smaller differences in SNGFR determined from paired proximal and distal tubular collections, and by smaller fractional changes of distal SNGFR in response to adding 6 nl/min of artificial tubular fluid to free-flowing proximal tubules. The TGF response in cd73(+/+) mice and the residual TGF response in cd73(-/-) mice were completely inhibited by adenosine A(1)-receptor blockade. The results suggest that extracellular formation of adenosine by ecto-5'-nucleotidase (cd73) is dispensable for normal fluid, Na(+), or K(+) reabsorption along the nephron, but contributes to the regulation of GFR. Adenosine generated by both ecto-5'-nucleotidase (cd73)-dependent and -independent mechanisms participates in the mediation of TGF in vivo.