AQP9 is an aquaglyceroporin that serves important functions in peripheral organs, including the liver. Reflecting the lack of AQP9 knockout mice, uncertainties still prevail regarding the localization and roles of AQP9 in the central nervous system. Here we present a comprehensive analysis of AQP9 gene expression in brain, based on a quantitative and multipronged approach that includes the use of animals with targeted deletion of the AQP9 gene. We show by real-time PCR that AQP9 mRNA concentration in rat and mouse brain is approximately 3% and approximately 0.5%, respectively, of that in rat and mouse liver, the organ with the highest level of AQP9. By blue native gel analysis it could be demonstrated that the brain contains tetrameric AQP9, corresponding to the functional form of AQP9. The band corresponding to the AQP9 tetramer was absent in AQP9 knockout brain and liver. Immunocytochemistry and in situ hybridization analyses with AQP9 knockout controls show that subpopulations of nigral neurons express AQP9 both at the mRNA and at the protein levels and that populations of cortical cells (including hilar neurons in the hippocampus) contain AQP9 mRNA but no detectable AQP9 immunosignal. The present data provide conclusive evidence for the presence of tetrameric AQP9 in brain and for the expression of AQP9 in neurons.