Three-dimensional cell cultures (spheroids) of biopsies of human duodenum were used to develop a new noninvasive method for studying intercellular and intracellular mechanisms. Through examinations of intracellular pH regulation, high functional similarity to native tissue could be shown, as already evidenced morphologically. A special microperfusion chamber was developed to fix individual spheroids physically to a nylon net, via laminar perfusion flow through the chamber. A significant improvement over current fixation methods was shown by the increase of cell viability almost up to 100%. Viability of the spheroids was confirmed by trypan blue exclusion, by a LIVE/DEAD viability/cytotoxicity kit, and by BCECF distribution. Intracellular pH was measured by use of the pH-sensitive fluorescence dye BCECF. To investigate the intracellular pH regulation, spheroid-like vesicles were acidified by NH₄Cl prepulse technique. The subsequent active intracellular pH recovery was blocked with Na -free Krebs Henseleit (KH) solution, with amiloride KH (inhibitor of the Na -H -exchanger), or with H₂DIDS KH (inhibitor of the HCO₃⁻-Cl⁻-exchanger and Na -HCO₃⁻-cotransporter). The intracellular pH of the spheroids was 7.31 ± 0.05. pH_i-backregulation after acidification was prevented by sodium-free buffer, amiloride, and H₂DIDS. These experiments indicated the presence of a Na -H -exchanger and a Na -HCO₃⁻-cotransporter. In conclusion, the human duodenal spheroid is an excellent physiological system for in vitro studies of the human duodenum.