The presence of Na(+)-H+ exchange activity is demonstrated on both the brush-border membrane (BBM) and the basolateral membrane (BLM) of villus cells from rabbit ileum. The possibility that the Na(+)-H+ exchange activity on the BLM represents HCO3- cotransport is excluded. The two Na(+)-H+ exchangers are then compared in terms of kinetics and substrate and inhibitor specificity. The most striking difference between the two exchangers was sensitivity to amiloride and K+. The IC50 for amiloride on the BLM was 10-fold lower than the BBM (11.2 +/- 2.1 vs. 103 +/- 20.9 microM; P less than 0.02). External K+, in concentrations as low as 10 mM, inhibited Na(+)-H+ exchange on the BBM but not on the BLM. The Na+ Km and proton Km were twice as high on the BLM exchanger (46.3 +/- 3.4 vs. 28.8 +/- 2.3 mM and 468 +/- 9 vs. 232 +/- 45 nM, respectively). Proton Vmax was similar, whereas Na+ Vmax was higher on the BLM. Inhibition by Li+ was similar on both membranes. These results indicate distinct differences between the two Na(+)-H+ exchangers. Whether these differences are due to the two different gene products or are the result of posttranslational modification of a single gene product remains to be determined.