Brush border vesicles were isolated from surgically resected pieces of human jejunum and ileum using a Mg2+/EGTA precipitation method. When compared to the homogenate, the final membrane preparation contained alkaline phosphatase at a 14 times higher concentration and almost no (Na++K+)-stimulated adenosine triphosphatase. An Na+/H+ antiport could be demonstrated in the jejunum by imposing a pH gradient between the interior and the outside of the vesicles (pHinside 5.2, pHoutside 7.2). In the presence of amiloride or harmaline, Na+/H+ antiport was inhibited by 60 +/- 5% (p less than 0.05) or 65 +/- 5% (p less than 0.05), respectively. In vesicles of human ileum we found an Na+/H+ antiport and in contrast to the jejunum a Cl-/OH- antiport could be demonstrated by imposing a pH gradient (pHinside 5.2, pHoutside 7.2). Besides this double-exchange mechanism for sodium and chloride, a Na+/Cl- cotransport and a Cl- conductive pathway could be detected in ileal brush border vesicles. In the presence of the anion transport inhibitors, furosemide, SITS and DIDS activities of Cl-/OH- antiport and Na+/Cl- cotransport were suppressed by 30 +/- 5% (p less than 0.05), 35 +/- 5% (p less than 0.05) and 40 +/- 5% (p less than 0.05), respectively. We conclude that absorption of sodium and chloride in the absence of organic solutes is mediated through different transport mechanisms at the luminal plasma membrane, which are in part subject to regulation by sodium and chloride transport inhibitors.