Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70: 69: 29 wild type: heterozygote: knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew; 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wildtype mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.