Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitous intracellular Ca2+ channels in the ER. Regulation of IP3Rs by Ca2+ allows regenerative propagation of Ca2+ signals, generating a hierarchy of Ca2+ release events. The spatial arrangement and dynamics of IP3Rs are important for producing these regenerative Ca2+ signals. To study the distribution and dynamics of IP3Rs at native expression levels, we used transcription activator-like effector nucleases (TALENs) to tag endogenous IP3R1 of HeLa cells with EGFP. EGFP-IP3Rs were functional and formed small clusters within ER membranes. Most IP3R clusters were mobile, but some were immobile for protracted periods. There was minimal mixing of the mobile and immobile populations of IP3Rs. Single-particle tracking revealed that IP3Rs move by diffusion, and along microtubules by both kinesin-1 and dynein motors. Within both mobile and immobile IP3R puncta, some IP3Rs were tightly packed but others were too far apart for their association to be mediated by direct interactions between IP3Rs. Simultaneous visualization of EGFP- IP3Rs and Ca2+ signals showed that Ca2+ signals, whether evoked by photolysis of caged IP3 or activation of endogenous receptors that stimulate IP3 formation, originate from immobile IP3Rs at ER-plasma membrane (PM) junctions. These Ca2+ release sites closely apposed the ER-PM junctions where stromal-interaction molecule (STIM), the ER Ca2+ sensor that stimulates store-operated Ca2+ entry (SOCE), accumulated after depletion of ER Ca2+ stores. Our results show that IP3-evoked Ca2+ signals are initiated by immobile IP3R clusters tethered near the ER-PM junctions at which SOCE occurs. We suggest that this organization may both optimize delivery of IP3 to IP3Rs and allow effective regulation of SOCE by local depletion of Ca2+ stores.