Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na⁺ absorption mediated by Na⁺/H⁺ exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na⁺ absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g. cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current, I_sc) across NHE3-null [NHE3(-)] murine small intestine and electroneutral Na⁺ absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway (CFTR-null [CFTR(-)] or Na⁺/K⁺/2Cl^- cotransporter-null [NKCC1(-)] mice). Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(-) as compared to wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation were significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(-) and NKCC1(-) intestine, Na⁺ absorption was significantly reduced as compared to wild-type. NHE3 protein expression was decreased in the CFTR(-) intestine, but unchanged in the NKCC1(-) intestine, indicating that factors independent of expression also down-regulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.