Calmodulin is thought to be the most important calcium sensor in signal transduction. When calcium binds to its 4 calcium binding sites, CaMCa4 becomes a multifunctional activator that regulates a large number of critical cell functions. In the nucleus, CaMCa4 controls gene transcription and other important nuclear processes. Here, using confocal microscopy to measure changes in fluorescence resonance energy transfer (FRET) of an indicator composed of two GFP-variants (CFP and YFP) linked by a calmodulin-binding sequence, we show that free cytosolic CaMCa4 concentration increased rapidly after a cell-wide increase in calcium concentration while the initial free CaMCa4 in the nucleus remained much lower and only equilibrated to the cytosolic CaMCa4 concentration after a delay of 1-2 minutes. Thus, immediately after a cell-wide calcium increase, a significant gradient in free CaMCa4 concentration exists between the cytosol and nucleus.