Aquaporin (AQP)-3 and AQP4 water channels are expressed at the basolateral membrane of mammalian collecting duct epithelium. To determine the contribution of AQP4 to water permeability in the initial inner medullary collecting duct (IMCD), osmotic water permeability (P _f) was compared in isolated perfused IMCD segments from wild-type and AQP4 knockout mice. The AQP4 knockout mice were previously found to have normal gross appearance, survival, growth, and kidney morphology and a mild urinary concentrating defect (T. Ma, B. Yang, A. Gillespie, E. J. Carlson, C. J. Epstein, and A. S. Verkman. J. Clin. Invest. 100: 957–962, 1997). TransepithelialP _f was measured in microdissected IMCDs after 18–48 h of water deprivation and in the presence of 0.1 nM arginine vasopressin (to make basolateralP _f rate limiting). P _fvalues (37°C; means ± SE in cm/s × 10⁻³) were 56.0 ± 8.5 for wild-type mice (n = 5) and 13.1 ± 3.7 for knockout mice (n = 6) (P < 0.001). Northern blot analysis of kidney showed that transcript expression of AQP1, AQP2, AQP3, and AQP6 were not affected by AQP4 deletion. Immunoblot analysis indicated no differences in protein expression of AQP1, AQP2, or AQP3, and immunoperoxidase showed no differences in staining patterns. Coexpression of AQP3 and AQP4 in Xenopus laevis oocytes showed additive water permeabilities, suggesting that AQP4 deletion does not affect AQP3 function. These results indicate that AQP4 is responsible for the majority of basolateral membrane water movement in IMCD but that its deletion is associated with a very mild defect in urinary concentrating ability.