Maintenance of ion balance requires that ionoregulatory epithelia modulate ion flux in response to internal or environmental osmotic challenges. We have explored the basis of this functional plasticity in the gills of the euryhaline killifish Fundulus heteroclitus. The expression patterns of several genes encoding ion transport proteins were quantified after transfer from near-isosmotic brackish water [10 parts/thousand (ppt)] to either freshwater (FW) or seawater (SW). Many changes in response to SW transfer were transient. Increased mRNA expression occurred 1 day after transfer for Na+-K+-ATPase-α1a(3-fold), Na+-K+-2Cl−-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3-fold) and was paralleled by elevated Na+-K+-ATPase activity (2-fold). The transient increase in NKCC1 mRNA expression was followed by a later 2-fold rise in NKCC protein abundance. In contrast to the other genes studied in the present work, mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel generally remained elevated (2-fold) in SW. No change in protein abundance was detected, however, suggesting posttranscriptional regulation. The responses to FW transfer were quite different from those to SW transfer. In particular, FW transfer increased Na+-K+-ATPase-α1amRNA expression and Na+-K+-ATPase activity to a greater extent than did SW transfer but had no effect on V-type H+-ATPase expression, supporting the current suggestion that killifish gills transport Na+via Na+/H+exchange. These findings demonstrate unique patterns of ion transporter expression in killifish gills after salinity transfer and illustrate important mechanisms of functional plasticity in ion-transporting epithelia.