Aquaporins are channel proteins widely expressed in nature and known to facilitate the rapid movement of water across numerous cell membranes. A mammalian aquaporin, AQPS, was recently discovered and found to have a very distinct evolutionary pathway. To understand the reason for this divergence, here we define the ontogeny and exact subcellular localization of AQPS in mouse liver, a representative organ transporting large volumes of water for secretion of bile. Northern blotting showed strong AQPS expression between fetal day 17 and birth as well as at weaning and thereafter. Interestingly, this pattern was confirmed by immunohistochemistry and coincided both temporally and spatially with that of hepatic glycogen accumulation. As seen by reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, fasting was accompanied by remarkable down-regulation of hepatic AQPS that paralleled the expected depletion of glycogen content. The level of hepatic AQPS returned to be considerable after refeeding. Immunoelectron microscopy confirmed AQPS in hepatocytes where labeling was over smooth endoplasmic reticulum (SER) membranes adjacent to glycogen granules and in canalicular membranes, subapical vesicles, and some mitochondria. In conclusion, in addition to supporting a role for AQPS in canalicular water secretion, these findings also suggest an intracellular involvement of AQPS in preserving cytoplasmic osmolality during glycogen metabolism and in maintaining mitochondrial volume. AQPS may have evolved separately to feature these intracellular roles as no other known aquaporin shows this specialization. (HEPATOLOGY 2003;38:947-957.) quaporins are a family of proteinaceous channels widely distributed in a range of living organisms A from bacteria to humans, where they mediate the osmotic movement of water across biological membranes.' Aquaporins are being shown to be involved in physiologic processes of central importance, including se- cretion and absorption of fluids in the gastrointestinal and reproductive tracts, renal water handling, fluid balance in the lung and brain, maintenance of fluid and ionic homeostases in the inner ear,2 and preserving corneal transparency.3 Aquaporins also have pathophysiologic relevance because they have been found in humans to underlie severe forms of nephrogenic diabetes in~ipidus,~ defective urinary concentrating,5 and congenital cataracts,6 and their involvement in the pathogenesis of many other clinical conditions with altered fluid homeostasis is under intense investigation. Among the 1 1 aquaporins (AQPO-AQP 10) that have so far been recognized in mammals, AQP8 represents a very distinctive homologue as indicated by the striking divergence of its evolutionary pathway.' However, the reasons for this phylogenetic divergence are obscure. AQP8 was recently cloned from rat,8,9 mouse,1° and human' ' tissues. By heterologous expression in Xenopw hevis oocytes, the rat8,9 and human' ' AQP8 channels were found to be selectively permeable to water, whereas the mouse...