The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V(te) and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V(te) and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine. It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.