Gametes of gray treefrogs, Dryophytes chrysoscelis, are deposited into freshwater ponds. Sperm undergo spermatogenesis and maturation beginning in the seminiferous tubules and migrating to the lumen. In mammals and fishes, these cells are immotile within the isosmotic fluid of the testes and have motility activated by exposure to a hyper- or hypoosmotic medium. Water flows into or out of the sperm cell, altering intracellular ionic concentrations, and ultimately stimulates flagellar movement. We tested the hypothesis that exposure to a hypotonic environment activates motility of gray treefrog sperm. We also hypothesized that osmotic water uptake is facilitated by expression of water channel proteins from the aquaporin family. To test these hypotheses, we collected sperm from captive treefrogs maintained with food and water at 22¿C and assessed motility of sperm immersed in hypoosmotic solutions of 200mOsm/L, 100mOsm/L, 75mOsm/L, 50mOsm/L, 25mOsm/L, and 10mOsm/L. A significant peak in reactivation percentages was seen at 50mOsmL (two-way ANOVA: P<0.0001, F(6,24)=20.64). Motility was scored based off of projectile patterns, and were denoted as forward moving, wobblers, circular movers, and tail movers. Osmotic activation had a significant effect on forward movers (One-way ANOVA: P<0.0001, F(6,28)=9.093), as well as circular movers (One-way ANOVA: P=0.0307, F(6,28)=2.766). We looked for the significance of temperature on motility activation and viability; cooler temperatures (4¿C) did not affect flagellar activation when compared to room temperature (22¿C). However, cooler temperatures did prolong viability over a period of 24h (two-way repeated measures ANOVA: P<0.0001, F(1,12)=568.7). We also assessed expression of mRNA and protein for two aquaporins, HC-1, a homolog of the water channel AQP1, and HC-7, a homolog of the glyceroporin (glycerol/water channel) AQP7, in sperm and testes from those warm-acclimated animals and from animals that were cold-acclimated during the autumn and winter. We detected mRNA via PCR for both HC-1 and HC-7 in testes from warm-acclimated, cold-acclimated, and post-freezing thawed animals, but not in emitted sperm. At the protein level, immunolocalization of HC-1 in a cross section of warm testes indicated protein expression in the mesentery surrounding each testis, in the epididymis, and in interstitial cells. No HC-1 expression was evident in the immature spermatogonia or in mature spermatozoa within the tubular lumen. HC-7 in warm testes was expressed in the interstitial tissues of the testes and, at low levels, in primary spermatocytes within the seminiferous tubules of testes. No HC-7 expression was detected in mature sperm cells. Western blot analysis concurred with both RNA and IHC results showing a presence of both HC-1 and HC-7 in liver and various testes conditions, however no protein was detected in ejaculated sperm. We conclude that treefrog sperm require osmotic activation to acquire motility. That water uptake likely is achieved via water channel proteins, but the specific aquaporins that are involved remains to be confirmed.