In teleost fish, prolactin (PRL) has important actions in the regulation of salt and water balances in freshwater (FW). Consistent with this role, the release of PRL from the pituitary of the Mozambique tilapia is stimulated as extracellular osmolality is reduced. Stretch-activated calcium-permeant ion channels appear to be responsible for the initiation of the signal transduction that leads to increased PRL release when PRL cells are exposed to reductions in extracellular osmolality. In this study, we examined a possible involvement of the aquaporin-3 (AQP3) water channel in this osmoreceptive mechanism in PRL cells of the tilapia. AQP3 expression levels in the rostral pars distalis of the pituitary, consisting predominantly of PRL cells, were higher in fish adapted to FW than in seawater (SW)-adapted fish. Immunohistochemical studies revealed that AQP3 is located in the cell membrane and peri-nuclear region of PRL cells, with more intense immuno-signals in PRL cells of FW-adapted fish than in those of SW fish. In FW PRL cells, the magnitude of hyposmoticity-induced cell volume increase was greater than seen in SW PRL cells. Mercury, a potent inhibitor of AQP3, inhibited hyposmoticity-induced cell volume increase and PRL release from FW PRL cells. The inhibitory effect of mercury was partially restored by -mercaptoethanol, whereas no effect of mercury was observed on PRL release stimulated by a depolarizing concentration of KCl, which induces Ca²⁺ influx and stimulates the subsequent Ca²⁺-signaling pathway. These results indicate significant contribution of AQP3 to osmoreception in of PRL cells in FW-adapted tilapia.