Genetically encoded Ca2+ indicators are outstanding tools for the assessment of intracellular/organelle Ca2+ dynamics. Basically, most indicators contain the Ca2+-binding site of a (mutated) cytosolic protein that interacts with its natural (mutated) interaction partner upon binding of Ca2+. Consequently, a change in the structure of the sensor occurs that, in turn, alters the fluorescent properties of the sensor. Herein, we present a new type of genetically encoded Ca2+ indicator for the endoplasmic reticulum (ER) (apoK1-er (W. F. Graier, K. Osibow, R. Malli, and G. M. Kostner, patent application number 05450006.1 at the European patent office)) that is based on a single kringle domain from apolipoprotein(a), which is flanked by yellow and cyan fluorescent protein at the 3'- and 5'-ends, respectively. Notably, apoK1-er does not interact with Ca2+ itself but serves as a substrate for calreticulin, the main constitutive Ca2+-binding protein in the ER. ApoK1-er assembles with calreticulin and the protein disulfide isomerase ERp57 and undergoes a conformational shift in a Ca2+-dependent manner that allows fluorescence resonance energy transfer between the two fluorophores. This construct primarily offers three major advantages compared with the already existing probes: (i) it resolves perfectly the physiological range of the free Ca2+ concentration in the ER, (ii) expression of apoK1-er does not affect the Ca2+ buffering capacity of the ER, and (iii) apoK1-er is not inactivated by binding of constitutive interaction partners that prevent Ca2+-dependent conformational changes. These unique characteristics of apoK1-er make this sensor particularly attractive for studies on ER Ca2+ signaling and dynamics in which alteration of Ca2+ fluctuations by expression of any additional Ca2+ buffer essentially has to be avoided.