Inositol trisphosphate receptors (IP3Rs) are Ca2+-permeable channels in the membrane of the endoplasmic reticulum (ER) that liberate Ca2+ sequestered in ER stores to generate cytosolic Ca2+ signals that control diverse cellular functions including gene expression, secretion and synaptic plasticity (Berridge et al., 2000). The spatial distribution of these channels is crucial in determining the patterning of intracellular Ca2+ signals. The mechanisms underlying the aggregation and maintenance of IP3Rs in clusters are controversial. Local Ca2+ signals arise at just a few, fixed locations within a cell, suggesting clusters are stable entities; and Ca2+ blips generated by ‘lone’ IP3Rs are also immotile (Smith et al., 2009). In contrast, GFP-tagged or immunostained IP3Rs show a dense distribution throughout a cell. Moreover, the majority IP3Rs can diffuse freely within the ER membrane, and aggregate into clusters following sustained (minutes) activation of IP3 signaling and/or cytosolic Ca2+ elevation, or even undergo clustering in response to IP3 within just a few seconds (Taufiq Ur et al., 2009). These apparently different behaviors may be explained because Ca2+ imaging studies detect only functional IP3Rs, whereas imaging studies utilizing immunostaining or GFP-tagged IP3Rs report on the entire population of IP3R proteins. We therefore hypothesize that a majority of IP3Rs are motile, but functionally unresponsive. Local Ca2+ signals arise, instead, from a small subset of IP3Rs that are anchored, individually or in clusters, by association with static cytoskeletal structures and possibly as a consequence of this anchoring, display high sensitivity to IP3 to Ca2+ signals. To test this hypothesis we have created a fusion protein of the IP3R tagged with the photoactivatable genetically encoded protein mEos2 to track the motility of thousands of single IP3Rs with nanoscale spatial and millisecond temporal resolution following transfection in cells (single-particle tracking photoactivated localization microscopy: sptPALM (Manley et al., 2008)). By tracking the movement of single IP3Rs using sptPALM we show that two populations of molecules exist with differing motilities. A larger (70%) of fast moving molecules (0.093 ± 0.009 μm2 s-1) and a smaller (30%) population of IP3Rs that are essentially immotile (0.0074 ± 0.0006 μm2 s-1, n > 25,000 tracks, 5 cells). Further, we find that these apparently immotile IP3Rs are preferentially grouped within tight clusters and may correspond to the static Ca2+ release sites responsible for generating local Ca2+ signals.