Glomerular tubular balance maintains a stable fractional solute and fluid reabsorption in the proximal tubule over a range of glomerular filtration rates. The mediators of this process are unknown. We tested the hypothesis that adenosine, produced in proximal tubule cells acting on adenosine type 1 receptors (A1-AR) promotes Na+and fluid uptake and mediates glomerular tubular balance. Absolute proximal fluid reabsorption ( Jv) was measured by in vivo microperfusion in A1-AR knockout and wild-type mice during perfusion of the closed proximal tubule at 2–10 nl/min. Jvincreased with perfusate flow from 2–4 nl/min in both strains, but the fractional increase was lower in A1-AR−/−mice (A1-AR+/+: 114% vs. A1-AR−/−: 38%; P < 0.001), suggesting reduced glomerular tubular balance (GTB). At higher perfusion rates, Jvincreased modestly in both strains, indicating less GTB at higher flow. The physiological effects of reduced GTB in A1-AR−/−mice were assessed from the response to an acute volume load (1 ml/2 min). Na+excretion and urine flow increased 76 and 73% more in A1-AR−/−mice than A1-AR+/+over the following 30 min, accompanied by a higher proximal tubule flow (A1-AR−/−: 6.9 ± 0.9 vs. A1-AR+/+: 5.2 ± 0.6 nl/min; P < 0.05). The expression of the sodium-hydrogen exchanger 3 and sodium phosphate cotransporter-2 were similar between strains. In conclusion, GTB is dependent on adenosine acting on type 1 receptors in the proximal tubule. This may contribute to acute changes in Na+and fluid reabsorption.