Calcium depletion of the endoplasmic reticulum (ER) induces oligomerisation, puncta formation and translocation of the ER Ca(2+) sensor proteins, STIM1 and -2 into plasma membrane (PM)-adjacent regions of the ER, where they activate the Orai1, -2 or -3 proteins present in the opposing PM. These proteins form ion channels through which store-operated Ca(2+) influx (SOC) occurs. Calcium ions exert negative feed-back on SOC. Here we examined whether subplasmalemmal mitochondria, which reduce this feed-back by Ca(2+) uptake, are located within or out of the high-Ca(2+) microdomains (HCMDs) formed between the ER and plasmalemmal Orai1 channels. For this purpose, COS-7 cells were cotransfected with Orai1, STIM1 labelled with YFP or mRFP and the mitochondrially targeted Ca(2+) sensitive fluorescent protein inverse-Pericam. Depletion of ER Ca(2+) with ATP+thapsigargin (in Ca(2+)-free medium) induced the appearance of STIM1 puncta in the < or =100 nm wide subplasmalemmal space, as examined with TIRF. Mitochondria were located either in the gaps between STIM1-tagged puncta or in remote, STIM1-free regions. After addition of Ca(2+) mitochondrial Ca(2+) concentration increased irrespective of the mitochondrion-STIM1 distance. These observations indicate that mitochondria are exposed to Ca(2+) diffused laterally from the HCMDs formed between the PM and the subplasmalemmal ER.