The aim of the present study was to examine the possibility that Cl- uptake into both villus and crypt epithelial cells of rat duodenum occurs via an electroneutral Na-K-Cl coupled-transport mechanism. Sheets of villus cells and whole crypts were isolated using a Ca2+ chelation technique combined with continuous vibration at low temperatures. Structurally intact, viable epithelia from defined regions along the villus-crypt axis were produced. Uptake of 86Rb+ (as a proxy for K+) into both villus and crypt cells appeared to depend on a coupled process, as evidenced by the inhibition of 86Rb+ uptake by bumetanide and by the removal of either Na+ or Cl- from the bathing media. We report an improved method of isolation of viable enterocytes from defined regions along the villus-crypt axis. We demonstrate the presence of Na-K-Cl cotransport in both villus and crypt duodenal enterocytes.