It is well known that Glucagon (Gl) is released after a high protein diet and participates in water excretion by the kidney, principally after a protein meal. To study this effect in in vitro perfused inner medullary collecting ducts (IMCD), the osmotic water permeability (Pfµm/s) at 37ºC and pH 7.4 in normal rat IMCDs (n=36) perfused with Ringer/HCO₃ was determined. Gl (10^-7M) in absence of Vasopressin (AVP), enhanced the Pf from 4.38±1.40 to 11.16±1.44 (p<0.01). Adding Gl 10^{- 8}, 10^-7 and 10^-6M, the Pf responded in a dose-dependent manner. The Protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His¹-[Glu⁹] glucagon (10^-7M), blocked the Gl stimulated Pf but not the Vasopressin (AVP) stimulated Pf. There occurred an additional partial effect between Gl and AVP. The cAMP level was enhanced from the control 1.24±0.39 fm/mg prot to 59.70±15.18 after Gl 10^-7M in an IMCD cell suspension. The immunoblotting studies indicated an increase in AQP2 protein abundance of 27% ( cont-100.0± 3.9 vs Gl 127.53, p= 0.0035) in membrane fractions extracted from IMCD tubule suspension, incubated with 10^-6M Gl. Our data showed that: 1) Gl increased water absorption in a dose-dependent manner; 2) The anti-Gl blocked the action of Gl but not the action of AVP; 3) Gl stimulated the cAMP generation; 4) Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl controls water absorption by utilizing a Gl receptor, rather than a AVP receptor, increasing the AQP2 protein expression.