Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.