The wall effective permeability of hydrophilic substances labeled with fluorescent dyes was evaluated in an isolated cannulated rat single lymph vessel through a videomicroscope system. Sodium fluorescein (NaFl, molecular weight 332) and FITC-dextrans (molecular weight 4400, 12000 and 71200) were administered into the intraluminal space of the lymph vessels and then excited by a Xenon lamp. Changes in the fluorescence intensity of the dyes were continuously measured by a silicon intensified target camera through appropriate filters. Net flux of each dye in the wall of lymph vessels was calculated by relationship between the fluorescence intensity and the concentration of the dyes. NaFl and FITC-dextran 4400 (4kD) in the intraluminal space of isolated rat lymph vessels significantly penetrated the wall of lymph vessels. FITC-dextran 12000 (12kD) in the intraluminal space of isolated rat lymph vessels slightly passed through the lymphatic wall, while FITC-dextran 71200 (70kD) did not penetrate the wall. Intraluminal pressure ranging from 4 to 8 cmH₂O did not significantly affect the net flux of dyes used in the present study. After administration of CHAPS into the lymph vessels, the net flux of FITC-dextran 4400 and 12000, but not 71200, augmented significantly. These results suggest that small molecular hydrophilic substances ( 4400) are permeable from the intraluminal to extraluminal space of isolated lymph vessels and that the endothelial cell surface structure may play a barrier role in the effective permeability of large molecular hydrophilic substances (4400 to 12000) through the wall of the lymph vessels.