Accurate measurements of Ca²⁺ inside organella are essential for a comprehensive analysis of the Ca²⁺ redistribution upon cell stimulation. The genetically-encoded Ca²⁺ indicators allow specific Ca²⁺ measurements within subcellular compartments. Aequorin-based Ca²⁺ chemiluminescence combine high specificity of targeting with large dynamic range from micromolar to milimolar levels and high signal-to-noise ratio. It has been shown that fusion of aequorin to the green fluorescence protein (GA) improves stability and increases light yield (Baubet et al, PNAS 97: 7260-5, 2000). Here we have used two different aequorin chimeras, one with GFP and the other with monomeric red fluorescence protein (mRFP) targeted to organella such as nucleus, mitochondria and endoplasmic reticulum. The aim was to monitor simultaneous and independently subcellular Ca²⁺ in the same cells by spectral separation of the red and green chemiluminescence. The correct expression of the fusion proteins was determined by western blotting and subcellular localization of the fluorescent indicators. Using this novel technology we find that Ca²⁺ transients in mitochondria and endoplasmic reticulum are different in size and kinetics as compared to cytosol and nucleus in stimulated HEK293 cells.