Cyclic AMP is a key mediator of collecting duct (CD) Na and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase 3 (AC3) partly mediates vasopressin actions; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 (CD AC3 KO). Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced angiotensin II, but not vasopressin, induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na or K excretion during a normal or high salt diet, while plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na and water handling as compared to CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na and water excretion in general or vasopressin regulation of CD function in particular.