The thickness of the unstirred water layer in in vivo-lavaged canine jejunum has been estimated by observations on the kinetics of entrance of [14C]inulin into the intervillus space (IVS) from the luminal fluid. Concentrations of the inulin in the IVS at three different levels (upper, 350 micrometers; middle, 250 micrometers; and lower, 250 micrometers) were determined as a function of duration of lavage. The concentrations rose slowly, indicating that there was little or no convective mixing of the fluid between the villi. After 1-2 h of lavage, mean concentrations in the IVS were three to seven times higher than in the lavage fluid, indicating that water absorption occurred from the IVS and that solvent drag as well as diffusion played a role in the entrance of inulin into the IVS. Because the concentration was always greatest at the uppermost level of the IVS, water absorption from the IVS must have been restricted to that level. Analysis of the data also required the inclusion of a small secretory stream (5% of the absorptive flow) from the crypts to explain the experimental observations. The results demonstrate that substances absorbed into the villus tips must penetrate an unstirred layer of 500-1,000 micrometers; for those absorbed into the lateral surfaces of the villi, an additional barrier of as much as 800 micrometers exists.