We have explored the molecular and physiological responses of the euryhaline killifish Fundulus heteroclitus to transfer from brackish water (10% seawater) to 100% seawater for 12 h, 3 days or 7 days. Plasma[Na+] and [Cl–] were unchanged after transfer, and plasma cortisol underwent a transient increase. Na+/K+-ATPase activity increased 1.5-fold in the gills and opercular epithelium at 7 days (significant in gills only), responses that were preceded by three- to fourfold increases in Na+/K+-ATPase α1a mRNA expression. Expression of Na+/K+/2Cl– cotransporter 1, cystic fibrosis transmembrane conductance regulator (CFTR)Cl– channel, Na+/H+-exchanger 3(significant in opercular epithelium only) and carbonic anhydrase II mRNA also increased two- to fourfold after transfer. Drinking rate increased over twofold after 12 h and remained elevated for at least 7 days. Surprisingly,net rates of water and ion absorption measured in vitro across isolated intestines decreased ∼50%, possibly due to reduced salt demands from the diet in seawater, but water absorption capacity still exceeded the drinking rate. Changes in bulk water absorption were well correlated with net ion absorption, and indicated that slightly hyperosmotic solutions (⩾298 mmol l–1) were transported. There were no reductions in unidirectional influx of Na+ from luminal to serosal fluid or intestinal Na+/K+-ATPase activity after transfer. Overall, our results indicate that gill and opercular epithelia function similarly at a molecular level in seawater, in contrast to their divergent function in freshwater, and reveal unexpected changes in intestinal function. As such they provide further insight into the mechanisms of euryhalinity in killifish.