Abstract Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca 2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca 2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca 2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca 2+] c), mitochondria ([Ca 2+] m) and ER ([Ca 2+] ER) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca 2+] were unaffected. By contrast, Ca 2+ influx elicited significantly smaller increases in [Ca 2+] c and [Ca 2+] m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca 2+ influx, but that the transfer of Ca 2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.