Changes in the concentration of cytoplasmic calcium, [Ca2+ ]cyt are central regulators in many cellular signal transduction pathways including many lipid-mediated regulatory networks. Given this central role that [Ca2+ ] has during plant growth, monitoring spatial and temporal [Ca2+ ] dynamics can reveal a critical component of cellular physiology. Here, we describe the measurement of [Ca2+ ]cyt in Arabidopsis root cells using plants expressing Yellow Cameleon 3.6 (YC 3.6). YC3.6 is a Ca2+ -sensitive biosensor where the intensity of its fluorescence resonance energy transfer (FRET) signal changes as the Ca2+ level within the cell rises and falls. The FRET from this calcium reporter can be visualized using confocal microscopy and the resultant images converted to a quantitative map of the levels of Ca2+ using an approach called ratio analysis.