Aquaporins (AQPs) are ubiquitously expressed proteinaceous water channels involved in numerous physiological processes including bile production and apoptosis. Aquaporins 8 and 9 are expressed in hepatocytes, and are down regulated in a rat model of hepatocellular carcinoma (HCC). This led me to hypothesize that decreased AQP 8/9 expression is a significant factor underlying increased resistance to apoptosis in HCC. To test this hypothesis I sought to identify mechanisms that regulate AQP expression and localization in HCC cells in vitro. Subsequently I determined expression of AQP 8/9 in a chemically induced model of HCC in mice in vivo, and in human HCC tissue. Finally I addressed the effect of increasing AQP expression in HCC cells on subsequent tumor cell proliferation in vivo. Cyclic adenosine monophosphate (cAMP) and interlukin-6 (IL-6) signaling are critical to AQP expression/localization in hepatocytes. Using the rat H4IIE HCC cell line, neither IL-6 nor cAMP significantly altered AQP8 or 9 expression in vitro, although IL6 significantly inhibited AQP8 membrane localization. However, pharmacological inhibition of cAMP signaling significantly affected AQP-dependent water movement in response to osmotic challenge. Analysis of a mouse-DEN model of HCC demonstrated increased AQP8 expression in the plasma membrane of hepatic foci. In contrast both AQP8 and 9 membrane localization were significantly decreased in HCC tissue (48 wks) compared to non-tumor liver (NTL). Similarly, in human HCC, AQP9 membrane localization was significantly decreased versus NTL, although neither AQP8 nor 9 expression correlated with known HCC risk factors. Finally, promoter-controlled AQP8 expression in a rat model of HCC significantly inhibited subsequent tumor growth in vivo. In conclusion, pathways that regulate AQP expression and localization in hepatocytes appear to be deregulated, to varying degrees, in HCC in vitro. However, decreased AQP membrane expression is a common feature in human and rodent models of HCC. Inhibition of HCC proliferation by over-expressing AQP8 raises the possibility of future therapeutic targets to slow or regress HCC progression in vivo.