The characteristics of [51Cr]EDTA, [3H]methoxyinulin ([3H]MI), [14C]polyethylene glycol-4000 ([14C]PEG), and [3H]mannitol as markers of the extracellular space (e.c.s.) of isolated mucosa from the rat small intestine have been examined. 2. Unidirectional transmural fluxes across the rat jejunum of [3H]MI, [14C]PEG and [3H]mannitol have been measured in the absence of glucose or in the presence of 28 mM glucose. 3. Assuming that [14C]PEG does not enter the cells, [51Cr]EDTA and [3H]mannitol seem to have access to approximately 50 and 90% of the intracellular water respectively. 4. The commercially availabel [3H]MI had access to a space which exceeded th [14C]PEG space by 10%. Upon purification by gel filtration the high molecular weight fraction of the [3H]MI provided estimates of the e.c.s. identical with the estimates obtained with [14C]PEG. 5. For all the markers used the e.c.s. estimates remained constant between the 40th and 80th min of incubation. 6. In the absence of glucose the transepithelial net fluxes of each of the different markers were zero. In the presence of 28 mM glucose the serosato-mucosa fluxes of all markers were dramatically increased. The ratio between the serosa-to-mucosa and the mucosa-to-serosa fluxes increased in the order [3H]mannitol greater than [3H]MI greater than [14C]PEG. 7. The effect of glucose on the flux ratio of the marker substances suggests that glucose-induced net water transport to the serosa side of the gut wall represents the difference between a transcellular net water transport to the serosal side and a significant paracellular net water transport through the lateral intercellular spaces to the mucosal solution.
