OBJECTIVE Three different Na+/H+ exchange isoforms have been localized in mammalian ileum: Na+/H+ exchange isoform 1 (NHE1) on the basolateral membrane and NHE2 and NHE3 on the brush border membrane. The aim of this study was to determine whether NHE3 is the predominant isoform serving basal ileal Na+ absorption. METHODS Immunocytochemistry was performed on canine ileum with specific polyclonal antibodies. Absorption studies (n = 58) were performed in dogs with 25-cm ileal Thiry-Vella fistulas. In groups 1-4, luminal 5-(N,N-dimethyl)-amiloride (DMA) was administered at doses of 100 nmol/L, 5 mumol/L, and 0.7 mmol/L to inhibit NHE1, NHE2, and NHE3, respectively. In groups 5-8, 1 mmol/L phlorizin (PHLZ), 0.7 mmol/L DMA, and 1 mmol/L PHLZ plus 0.7 mmol/L DMA were administered intraluminally. RESULTS NHE2 and NHE3 were present in the brush border of canine ileal villus epithelial cells. The highest concentration of DMA (0.7 mmol/L) caused a significant reduction (P < 0.05) in basal ileal water and sodium absorption. PHLZ and 0.7 mmol/L DMA each alone significantly reduced basal ileal absorption (P < 0.05), whereas PHLZ plus 0.7 mmol/L DMA had an additive effect. CONCLUSIONS NHE3 seems to be the Na+/H+ exchange isoform involved in the modulation of basal ileal water and sodium absorption. Both Na+/H+ exchange mediated through NHE3 and Na(+)-glucose cotransport contribute to basal ileal water and sodium absorption.