MDCK I and Fisher rat thyroid cells exhibit transepithelial electrical resistance (TER¹) values in excess of 5,000 -cm². When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a greater than 5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line which typically exhibits values below 800 -cm². These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.