Cotton (Gossypium hirsutum), the most important textile crop worldwide, often encounters water stress such as drought or waterlog during its growth season (Summer). To investigate molecular mechanism of water regulation in cotton plants, three cDNAs encoding the plasma membrane intrinsic protein (PIP) were isolated from cotton root cDNA library, and designated GhPIP1;1, GhPIP2;1 and GhPIP2;2, respectively. All of the three PIP proteins displayed water channel activity in Xenopus laevis oocytes. GhPIP2;1 and GhPIP2;2 proteins, however, showed much higher water transport activity than that of the GhPIP1;1 protein. Northern blot analysis revealed that all of the three genes were preferentially expressed in young roots. Further analysis by Real-time quantitative RT-PCR revealed that the transcripts of all the three genes were accumulated at high levels in 3-day-old young roots, but dramatically declined to much lower levels in 6-14 days old roots during seedling development, suggesting that expressions of the isolated GhPIP genes are developmentally regulated in roots. Additionally, expressions of the three genes were remarkably up-regulated or down-regulated under different stresses such as NaCl, cold, PEG (polyethylene glycol) treatments. Collectively, the results suggest that these genes may be involved in root development and in response to stresses.