We identified and investigated the changes in expression of two gill Na+, K+ -ATPase alpha-subunit isoforms (alpha-1a and alpha-1b) in relationship with salinity acclimation in a cichlid fish, Mozambique tilapia. Transfer of freshwater (FW)-acclimated fish to seawater (SW) resulted in a marked reduction in alpha-1a expression within 24 h and a significant increase in alpha-1b expression with maximum levels attained 7 days after the transfer. In contrast, transfer of SW-acclimated fish to FW induced a marked increase in alpha-1a expression within 2 days, while alpha-1b expression decreased significantly after 14 days. Hypophysectomy resulted in a virtual shutdown of alpha-1a mRNA expression in both FW- and SW-acclimated fish, whereas no significant effect was observed in alpha-1b expression. Replacement therapy by ovine prolactin (oPrl) fully restored alpha-1a expression in FW-acclimated fish, while cortisol had a modest, but significant, stimulatory effect on alpha-1a expression. In hypophysectomized fish in SW, replacement therapy with oPrl alone or in combination with cortisol resulted in a marked increase in alpha-1amRNA to levels far exceeding those observed in sham-operated fish. Expression of alpha-1b mRNA was unaffected by hormone treatment either in FW-acclimated fish or in SW-acclimated fish. The mRNA expression of fxyd-11, a regulatory Na+, K+ -ATPase subunit, was transiently enhanced during both FW and SW acclimation. In hypophysectomized fish in FW, oPrl and cortisol stimulated fxyd-11 expression in a synergistic manner. The clear Prl dependence of gill alpha-1a expression may partially explain the importance of this hormone to hyperosmoregulation in this species. Journal of Endocrinology (2011) 209, 237-244