To examine directly in real time the efflux of organic compounds [e.g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K_t^lumen) of ~560 µM and maximal rate of FL efflux across the luminal membrane (J_max^lumen) of ~635 fmol · min¹ · mm¹]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.