1. To test whether colonic crypts are secretory or absorptive interstitial [Na+] in rat descending colonic mucosa is determined using video-enhanced imaging of the impermeant acid form of the fluorescent Na+ probe SBFI (Molecular Probes) and intracellular [Na+] is monitored with SBFI (AM form). In rat descending colonic mucosa perifused with isotonic Tyrode solution interstitial [Na+] = 500-650 mM. Following exposure to Tyrode solution containing theophylline (10 mM) interstitial [Na+] falls by 300-450 mM within 1 min. Exposure to amiloride (0.2 mM) reduces the intracellular [Na+] from ca 25 to 12 mM within 15 min and concurrently decreases [Na+] in the interstitial fluid surrounding the crypts at the mucosal surface by approximately 200 mM. 2. The route of fluid inflow across the rat colonic mucosa is directly traced by perifusing with Tyrode solution containing the impermeant fluorescent dye, fluorescein disulphonate (FS). FS accumulates rapidly within crypt lumens of control tissues to a 2-fold higher concentration than in the external bathing solution, but FS does not accumulate in crypts of tissues treated with azide (2 mM). The increment in FS accumulation within the crypt lumen above the bulk solution decreases by 80% within 1 min following exposure to theophylline (10 mM), indicating that fluid absorption into crypts is reduced. Estimates of the total fluid influx from the rate and extent of FS concentration polarization within crypts indicate that it is sufficient to account for the entire transcolonic fluid absorption. 3. Comparative studies of isolated bovine and ovine colon were also undertaken to investigate the failure of bovine colon to generate a hypertonic absorbate and hence its incapacity to produce hard faeces. The interstitial fluid surrounding ovine colonic crypts is hypertonic to the bulk solution, whereas the interstitial fluid surrounding bovine colonic crypts is nearly isotonic with the bathing solution. Additionally, fluorescein disulphonate accumulates within ovine colonic crypt lumens by concentration polarization, whereas no concentration of FS occurs within bovine colonic crypt lumens. This corroborates the view that a hypertonic interstitial fluid is absent from bovine colon mainly because of a high rate of transepithelial leakage of low molecular weight solutes via paracellular routes.