Cope's gray treefrogs, Hyla chrysoscelis, accumulate glycerol during cold acclimation. We hypothesized that during this process, gray treefrogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200 fold, to 51 mmol/l. Although fractional water reabsorption decreased, GFR and, consequently, urine flow were less than 5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, was expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3 is most similar to AQP3 and is functionally characterized as a GLP, with permeability low to water but high to glycerol. Aspects of both expression levels and functional characteristics varied in cold vs. warm conditions for each of the three aquaporins, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.