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To characterize diarrhoeal mechanisms in HIV-infected patients, epithelial transport and barrier function of the duodenal mucosa was investigated in vitro.Twenty-one HIV-seropositive patients (13 asymptomatic and eight with diarrhoea) and 12 controls from an urban referral-based tertiary care centre in Berlin who underwent duodenoscopy.A new miniaturized Ussing chamber allowed measurements on duodenal forceps biopsies. Epithelial barrier function was characterized by alternating current impedance analysis, which allows differentiation of epithelial and subepithelial resistance and by 3H-lactulose and 3H-mannitol flux measurements. Na+-glucose cotransport was quantified as phlorizin-sensitive short circuit current (ISC) and active ion secretion by baseline and bumetanide-sensitive ISC.Duodenal biopsies from asymptomatic HIV-infected patients were no different from controls, whereas biopsies from HIV-infected patients with diarrhoea showed a decrease in epithelial resistance from 21.2 ± 1.9 to 12.9 ± 1.3 Ωcm2 (P < 0.01). Concomitantly, mucosal-to-serosal lactulose flux increased from 0.29 ± 0.02 to 0.40 ± 0.03 µmol (hcm2) (P < 0.01). Phlorizin-sensitive ISC indicating Na+-glucose cotransport, as well as baseline and bumetanide-sensitive ISC indicating active electrogenic chloride secretion were not different between the three groups.A miniaturized Ussing device was developed for electrophysiological investigations of duodenal forceps biopsies, which allowed characterization of active ion transport mechanisms and epithelial barrier function. Duodenum of HIV-infected patients with diarrhoea showed no evidence for active ion secretion or Na+-glucose malabsorption, but showed an impaired epithelial barrier function, which could contribute to diarrhoea by a leak flux mechanism.