Stromal interaction molecule 1 (STIM1) is responsible for activating the Ca(2+) release-activated Ca(2+) (CRAC) channel by first sensing the changes in Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)) via its luminal canonical EF-hand motif and subsequently oligomerizing to interact with the CRAC channel pore-forming subunit Orai1. In this work, we applied a grafting approach to obtain the intrinsic metal-binding affinity of the isolated EF-hand of STIM1, and further investigated its oligomeric state using pulsed-field gradient NMR and size-exclusion chromatography. The canonical EF-hand bound Ca(2+) with a dissociation constant at a level comparable with [Ca(2+)](ER) (512 +/- 15 microm). The binding of Ca(2+) resulted in a more compact conformation of the engineered protein. Our results also showed that D to A mutations at Ca(2+)-coordinating loop positions 1 and 3 of the EF-hand from STIM1 led to a 15-fold decrease in the metal-binding affinity, which explains why this mutant was insensitive to changes in Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)) and resulted in constitutive punctae formation and Ca(2+) influx. In addition, the grafted single EF-hand motif formed a dimer regardless of the presence of Ca(2+), which conforms to the EF-hand paring paradigm. These data indicate that the STIM1 canonical EF-hand motif tends to dimerize for functionality in solution and is responsible for sensing changes in [Ca(2+)](ER).