The adenylyl cyclase isoform 6 (AC6) is expressed in all renal tubular segments and the collecting duct (CD) and catalyzes the synthesis of cAMP. To define the in vivo role of this enzyme in arginine-vasopressin (AVP)-induced urine concentration, we studied AC6 wild-type (WT, n=8) and knockout (AC6^-/-, n=8) mice. With free access to food and water AC6^-/-, compared to WT, have higher fluid intake (13.50.5 vs. 5.00.3 ml/day, P<0.01), lower urine osmolality (Uosm: 87594 vs. 2027160 mmol/kg, P<0.01) but similar GFR (10.00.6 vs. 11.41.1 ml/min/g bw) and plasma osmolality (Posm: 3061 vs. 3052 mmol/kg), indicating intact fluid balance. 24 h water restriction revealed a lower Uosm in AC6^-/- vs. WT (2577110 vs. 309598 mmol/kg, P<0.01) in spite of a greater urinary AVP/creatinine ratio (112024 vs. 40571 nmol/mmol, P<0.05), associated with greater body weight loss (-4.30.5 vs. -2.80.1 g, P<0.05) and greater Posm (3393 vs. 3232 mmol/kg, P<0.01). Oral water-loading (3% of bw; to suppress endogenous AVP) lowered urine osmolality to similar levels in AC6^-/- vs. WT (25938 vs. 31763 mmol/kg) but total urinary cAMP excretion and the increase in Uosm with co-administration of the agonist dDAVP (0.1 mg/kg i.p.) were lower in AC6^-/- (1.00.1 vs. 2.70.7 pmol/min/g bw, P<0.05; 202061 vs. 312794 mmol/kg, P<0.01). These results demonstrate that expression of AC6 is important for urinary concentration in vivo, including in response to water restriction and AVP administration.