The NF-kB transcription factor family plays key regulatory roles in a number of critical cellular processes such as inflammation, the cell cycle and apoptosis. It is becoming clear that there is an extremely complex system of spatial and temporal regulation of this pathway which we are still yet to fully understand. Here it is illustrated how regulation of the nuclear translocation of the transcription factor NF-kB (p65) is tightly controlled by a mechanism involving a number of other proteins in the NF-kB signalling system. These control systems are differentially selected for use by the cell in order to allow stimulus-specific regulation of the nuclear translocation of the transcription factor. Single cell confocal microscopy analysis in particular has been used to examine the stimulus dependence of NF-kB transcription factor dynamics and to measure any signal-specific effects of other proteins on these dynamics.