Studies on transcellular transport across epithelial cell layers are performed mostly by discontinuous sampling of the transported compound. This has several drawbacks, e.g., it gives disturbances in volume, it limits the time-resolution, and is often laborious. In this report we introduce a method to measure transepi-thelial transport of fluorescent compounds continuously. The time-resolution is at the (sub)minute scale, allowing the measurement of the change in transport rate before and after transport modulation. We will describe how we used the method to measure transcellular and paracellular transport. For highly membrane-impermeable compounds, the paracellular transport and the regulation of the tight junctions was studied in wild-type and MDR1 cDNA transfected epithelial canine kidney cells (MDCKII). The effect of the multidrug transporter P-glycoprotein (Pgp) on the transepithelial transport was studied. Addition of the Pgp inhibitor SDZ PSC 833 showed a modulation of the idarubicin (IDA) and daunorubicin (DNR) transport, which was larger during transport from the basolateral to the apical side than in the reverse direction. By modeling the transepithelial transport, we found that in these cells Pgp had more effect on the basolateral to apical transport than vice versa, which can be attributed to a relatively large passive permeation coefficient for the cellular basolateral plasma membrane.