Epidermal growth factor (EGF) is well known to promote intestinal epithelial barrier function [1]. However, its role in regulating epithelial transport function is less well understood. We have previously shown that acute treatment with EGF chronically enhances epithelial responsiveness to both Ca²⁺ and cAMP-dependent secretagogues. Here, we investigated the molecular mechanisms involved. T₈₄ colonic epithelial cells were grown as polarized monolayers on permeable supports. Changes in I_sc, indicative of electrogenic ion transport, were measured by the Ussing chamber/voltage clamp technique. Protein and mRNA expression were measured by Western blotting and PCR, respectively. Intracellular Ca²⁺ ([Ca²⁺i]) was measured by Fura-2 fluorescence and protein kinase A (PKA) activity with the Promega PepTag assay kit. Acute treatment of T₈₄ cells with EGF (100 ng/ml; 15 min) chronically enhanced subsequent I_sc responses to the Ca²⁺-dependent agonist, carbachol (CCh, 100 µM). This effect was only apparent after 3 h and was maximal by 6 h after stimulation with EGF at which time responses to CCh were 206.1 ± 28.3% of those in control cells (n = 4; p< 0.01). The potentiating effect of EGF was sustained for at least 24 h. The Na⁺-K⁺-2Cl^– co-transporter (NKCC1) inhibitor, bumetanide (100 µM), abolished the effect of EGF, indicating Cl^– secretion to be involved. Neither basal nor agonist-stimulated levels of intracellular Ca²⁺ or PKA activity were altered by EGF pretreatment, implying the effects of the growth factor are not due to chronic alterations in levels of second messengers. Western blot analysis revealed that EGF increased the expression of NKCC1 with a time course that closely paralleled its effects on Cl^– secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 ± 21.9% of that in control cells (n =21; p<0.0001). EGF also increased the CFTR expression (by 3.9 ± 1.1-fold; n=5; p<0.01) but not that of the Na⁺/K⁺ ATPase pump. EGF-induced NKCC1 expression was abolished by the inhibitor of transcription, actinomycin D, implying EGF upregulates NKCC1 expression at the level of gene transcription. Finally, rtPCR analysis showed that within 1–4 h after stimulation with the growth factor, EGF increased expression of NKCC1 mRNA to levels that were 160 ± 11.9% of those in control cells (n=3; p<0.05). These data reveal a novel role for EGF in chronically regulating epithelial secretion at the fundamental level of transport protein expression. EGF enhances epithelial secretory capacity by inducing NKCC1 expression, an effect which in vivo would serve to promote epithelial hydration, thereby enhancing barrier function. These data suggest that further elucidation of EGF-dependent signalling pathways may yield new targets for drug development in treatment of intestinal transport disorders.