A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring colonic crypt function in vivo in mice. Na(+) concentration in the extracellular pericryptal space of descending colon was measured using a low-affinity Na(+)-sensitive fluorescent indicator consisting of an Na(+)-sensitive chromophore (sodium red) and an Na(+)-insensitive chromophore (Bodipy-fl) immobilized on 200-nm-diameter polystyrene beads. The Na(+) indicator beads accumulated in the pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na(+) concentration ([Na(+)]) in the pericryptal space was 491 +/- 62 mM (n = 4). After a 70-min exposure to amiloride (0.25 mM), pericryptal [Na(+)] was reduced to 152 +/- 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [Na(+)] to 204 +/- 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dye into the crypt lumen with a half time of 19.8 +/- 1.0 s, which was increased to 77.9 +/- 6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measure colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The pericryptal space represents the first example of a hypertonic extracellular compartment in mammals that is not created by a countercurrent amplification mechanism.