The Ussing chamber is increasingly being used for in vitro studies of human intestinal mucosa, but little attention has been paid to the viability of specimens over time. Ninety-one mucosal specimens from the ileum in 19 patients operated on for colonic cancer were studied in regard to intestinal barrier function, metabolism, electrophysiology and histology during 360 min of incubation in Ussing chambers. Steady-state permeability to 51Cr-EDTA was maintained for 120 min. Mucosal ATP and lactate levels were stable for 180 min and transmucosal glucose flux for 240 min. Lactate dehydrogenase leakage was limited within 120 min. Transepithelial potential difference was 9.0 +/- 3.0 mV at the start, and declined slowly throughout 360 min. Light microscopy revealed epithelial lifting from the basal lamina at 90 min. Transmission electron microscopy demonstrated preserved ultrastructure for 120 min. Specimens with a transepithelial potential difference below 6 mV at the start were associated with increased 51Cr-EDTA permeability and lactate dehydrogenase leakage and more pronounced light microscopy changes. All studied parameters pointed to preserved viability if experiments were kept within a period of 90 min after equilibration. The few specimens with early viability derangement were identified by a transepithelial potential difference below 6 mV at the start. The Ussing chamber provides a tool for in vitro studies of human intestinal epithelium, including permeability. To minimize viability problems, experiments should be limited in time and monitored by measurements of transepithelial potential difference.