Sodium/proton exchangers [Na⁺/H⁺ (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na⁺ and Cl⁻ flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(−)], and NHE3 knockout [NHE3(−)] mice. Under basal conditions, NHE(+) and NHE2(−) jejuna had similar rates of net Na⁺ (∼6 μeq/cm² · h) and Cl⁻ (∼3 μeq/cm² · h) absorption. In contrast, NHE3(−) jejuna had reduced net Na⁺ absorption (∼2 μeq/cm² · h) but absorbed Cl⁻ at rates similar to NHE(+) and NHE2(−) jejuna. Treatment with 100 μM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na⁺ and Cl⁻absorption in all genotypes. Studies of the Na⁺ absorptive flux (J ) indicated thatJ in NHE(+) jejunum was not sensitive to 1 μM EIPA, whereasJ in NHE3(−) jejunum was equally sensitive to 1 and 100 μM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP_i) abolished net NaCl absorption and stimulated electrogenic Cl⁻ secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(−) and NHE3(−) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na⁺ absorption;2) an amiloride-sensitive Na⁺ transporter partially compensates for Na⁺ absorption in NHE3(−) jejunum; 3) cAMP_i stimulation abolishes net Na⁺ absorption in NHE(+), NHE2(−), and NHE3(−) jejunum; and 4) electroneutral Cl⁻ absorption is not directly dependent on either NHE2 or NHE3.