Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties;Rmvalues and the partition coefficientsPo/wbetweenn-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dyeCDcan be increased fromCD=0 toCD>0 within a few seconds (concentration jump). Thus, the timet=0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment,CD=const., are unambiguously defined. The concentration of the dye,Cb, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescenceIb. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence,If≪Ib. The intensity of the fluorescenceI=Ibwas measured as a function of timet; the measured values were corrected for photobleaching. The fluorescence intensityI(t) at first increases linearly withtand reaches a saturation value fort→ ∞. In the linear range ofI(t) the flowJo=(dI/dt)oof the dye into the cell depends strongly on the dye concentration and increases linearly withCD. The concentration rangeCD=10−9−10−5Mat 37° C was investigated. From the linear correlation betweenJoandCDit follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. Att=0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slopedJo/dCDof the plotJovsCDdescribes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophil