CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-stimulated chloride channel which is located in the apical membrane of various epithelial cells. Upon stimulation CFTR is not only activated but also its plasma membrane density increases due to the recruitment from submembrane stores. Furthermore, CFTR exists in macromolecular complexes in the plasma membrane. However, it is yet unclear whether clustering is a result of stimulation. Therefore we investigated the rearrangement of CFTR molecules upon stimulation using immunostaining with quantum dot (QDot) labelled antibodies. These nanocrystaline fluorophores resist bleaching and they blink. Blinking analysis allows to distinguish between QDots located in close vicinity, far below the resolution of fluorescence microscopy. About 1000 images with 10 ms exposure were made and the intensity of single light spots was plotted over time. A single QDot shows two intensity levels (on/off), two QDots show three intensity levels (off/off, on/off, on/on). Using single ion channel recording software we determined the number of intensity levels. We performed immunostaining of CFTR on paraformaldehyde fixed Calu-3 cells with an antibody directed to an extracellular CFTR loop without and after stimulation with cAMP. Analysis of QDot blinking reveals that CFTR molecules exists unclustered in non-stimulated cells whereas CFTR clusters are detected in plasma membrane after stimulation. We conclude that stimulation of Calu-3 cells with cAMP induces clustering of CFTR