Objective: Aquaporin 9 (AQP9), in hepatocytes, is expressed in the sinusoidal membrane. In our hands mouse AQP9 is permeable to water and solutes including glycerol and urea. Furthermore, AQP9 ammonia permeability has been suggested previously. The role of AQP9 in the liver is not fully characterized. AQP9's localization in the sinusoidal membrane, suggests a function in hepatocyte ammonia/urea uptake/release. In the presented study, the physiological relevance of an AQP9 role in ammonia and/or urea transmembrane exchange in the liver was examined in wild type (WT) and AQP9-knockout (KO) mice. Methods: Since ammonia and urea are products of amino acid metabolism we exposed the hepatic system in two experimental paradigms to (I) short term or (II) long term intake of high amounts of amino acids. In paradigm I we forced fed AQP9 WT and KO mice with 2mmol/100 g body weight glutamine. In paradigm II WT and KO mice were fed on high protein diet for four days. Results: Single administration of glutamine with gavage (I) evoked elevation of ammonia. No significant differences in plasma, liver, and brain concentration of ammonia or urea were detected between knockout and wild type mice. In paradigm II we detected a significant increase of ammonia and urea level in urine in both AQP9 WT and KO mice but no significant difference in ammonia and urea levels in the plasma, liver, and urine between the genotypes. Conclusions: AQP9 is not essential for ammonia/urea exchange at the hepatocyte sinusoidal membrane. Robust alternative routes for uptake/release of these metabolites must exist.