Ca²⁺ indicators are powerful tools for the measurement of physiological signaling processes. However, their biochemical properties limit their targeting to subcellular structures such as the endoplasmic reticulum (ER), a major intracellular Ca²⁺ store. Here, we describe a new method for the efficient targeting of low affinity, acetoxymethyl (AM) ester-based Ca²⁺ indicators to the ER, thus substantially improving the signal quality of ER Ca²⁺ measurements. We found the overexpression of an ER-targeted carboxylesterase (CES) to be a suitable tool for the enhancement of ER loading with AM ester dyes. In various cell types, CES expression was validated by Western analysis and immunostaining. The expression of a GFP-CES-fusion protein confirmed the highly localized retention of these proteins in the ER. In CES-expressing BHK21 and HEK293 cells, ATP-induced and/or CPA(cyclopiazonic acid)-induced ER-specific Ca²⁺ depletion signals were measured with a good signal-to-noise ratio, when the low affinity calcium indicator Fluo5N-AM was used. In neurons, lentivirus-based expression of CES allowed the direct recording of metabotropic glutamate receptor-induced Ca²⁺ release. Additionally, local CPA-induced Ca²⁺ depletion of the ER could be observed in peripheral dendrites of cultured neurons. Thus, our results establish CES-targeting as a convenient tool for the analysis of ER calcium signaling.