BACKGROUND Duodenal mucosal bicarbonate secretion is an important first line of defense against gastric acid. Studies in the ileum indicate that the secretion originates from the crypt cells, whereas villus cells are mainly absorptive. Data on acid/base transporters along the crypt-villus axis in duodenal epithelia are not available. It was our purpose to identify and compare acid/base transporters in isolated mammalian duodenal villus and crypt cells. METHODS The proximal duodenum of rabbits was excised, and duodenal epithelial cells were isolated in five fractions by a modified calcium chelation technique. Intracellular pH (pHi) was measured with a pH-sensitive dye and dynamic fluorescence ratio imaging. RESULTS In both villus and crypt cells incubated in Hepes buffer, removal of Na+ or addition of amiloride decreased basal pHi and pHi recovery after intracellular acidification, indicating an Na+/H+ exchanger in both cell types. In both cell types acid extrusion rates in bicarbonate-buffered Ringer's solution were significantly higher than in Hepes buffer. The bicarbonate-dependent acid extruder was unaffected by removal of Cl- or addition of amiloride but was blocked by removal of Na+, indicating the presence of a NaHCO3 cotransporter in both villus and crypt cells. Removal of external Cl induced a reversible increase in pHi (inhibited by H2DIDS) in both villus and crypt cells, indicating a Cl-/HCO3- exchanger in both. CONCLUSIONS Mammalian duodenal villus and crypt cells have identical acid-base transporters. These findings tend to negate the theory of a functional difference in acid-base transporters between duodenal villus and crypt cells and instead imply alkaline secretion by both cell fractions. However, as these experiments were performed in unpolarized, single cells, additional studies with either membrane vesicles or polarized cells are needed.