A novel setup for fluorescence intensity and lifetime imaging (FLIM) of living cells is reported. Time-resolving techniques are combined with total internal reflection fluorescence microscopy (TIRFM), which permits optical excitation of either plasma membranes or whole cells depending on whether the angle of incidence of the excitation light is greater or smaller than the critical angle for total internal reflection. The method is applied to BKEz-7 endothelial cells incubated with various concentrations of the well established mitochondrial marker rhodamine 123(R123). Measurements show that only at low concentrations this dye is mainly located within the mitochondria, whereas at higher concentrations an accumulation within the plasma membrane occurs as well. Concomitantly, fluorescence quenching in the mitochondria is observed at high concentrations, probably due to aggregation of the R123 molecules. Therefore, for diagnostic applications the concentration of R123 in the incubation medium should not be above 25 microM.