A new method is presented for measuring rapid efflux of fluorescent compounds from monolayer cells. Cells grown on a glass coverslip were loaded with a fluorescent substrate. Thereafter, the coverslip was installed outside the light path in a stirred and thermostated cuvette of a fluorometer. The efflux was recorded by measuring the changes of fluorescence in the extracellular medium. The method was used to study the kinetics of active and passive plasma membrane transport of the P-glycoprotein substrates rhodamine 123 and daunorubicin. The method has advantages over other methods: (1) no radioactively labeled substrate is needed, (2) fluorescence of the transported substrate is not compromised by the cells, (3) changes in the extracellular concentration of the substrate can be monitored continuously and therefore a substantial improvement of the kinetic resolution is obtained, and (4) the measurement setup is relatively simple and a standard fluorometer can be used. From the efflux data, cellular transport parameters could be calculated, such as passive permeation coefficients and active transport rates.