In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, we cloned a cDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence of H. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conserved Asn-Pro-Ala motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevis oocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation, confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serum and tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed after freezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernating frogs. Glycerol content in the liver did not change in the freezing experiment, whereas that in the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a cryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells.