For the analysis of Ca(2+)-dependent signaling, acetoxymethyl (AM)-derivatized ion indicators have become a popular tool. These indicators permeate membranes in an ion-insensitive form but, within cells, esterases hydrolyze these compounds to release ion-sensitive dyes. However, the properties of these indicators Limit their targeting to subcellular structures such as the endoplasmic reticulum, the dominant intracellular Ca2+ store. This study presents a novel approach for trapping fluorescent Ca2+ indicators in the ER. The method combines the selectivity of protein targeting with the biochemical advantages of synthetic Ca2+ indicators and allows direct, non-disruptive measurements of Ca(2+)-store dynamics with a high structural and temporal resolution. A recombinant carboxylesterase was targeted to the ER, providing a local esterase activity. After esterase-based dye loading, this additional esterase activity allowed improved trapping of Ca(2+)-sensitive forms of low-affinity Ca2+ indicators (e.g. Fluo5N) within the ER. The utility of the method was confirmed using different cell systems (293T, BHK21, cortical neurons) and activating different signaling pathways. In neurons, this approach enabled the detection of ER Ca2+ release with high resolution. In addition, the method allowed rapid confocal imaging of Ca2+ release from the ER, after activation of metabotropic glutamate receptors, in the presence of extracellular Ca2+.