We have investigated the effects of all-trans retinoic acid (ATRA) on aquaporin 3 (AQP3) expression and function both in vitro and ex vivo. ATRA treatment provoked a rapid accumulation of AQP3 transcripts in cultured normal human epidermal keratinocytes (NHEK). This increase was still observed 24 hours after application of ATRA. The induction of AQP3 gene was accompanied by an augmentation of immunoreactivity. Using a selective agonist, we demonstrated that the effect of ATRA was predominantly mediated by retinoic acid receptor subtype gamma (RARgamma). Incubation of NHEK in ATRA for 24, 48, and 72 hours stimulated glycerol influx, suggesting that the increase in AQP3 gene and protein expression was followed by an enhancement of biological activity. Topical application of ATRA for 24 hours on skin explants induced significant epidermal expression of AQP3 and strong immunoreactivity in the epidermal basal layers. Collectively, the present results show that ATRA increased AQP3 expression and enhanced biological activity in human skin.