The uptake of calcium by mitochondria has traditionally been regarded as a pathological rather than a physiological phenomenon. However, recent work from a number of laboratories demonstrates that mitochondria will accumulate calcium during normal cell signalling processes. We have combined the whole cell patch clamp and confocal imaging techniques to study calcium dynamics in sensory neurones isolated from humanely killed E12 chick embryos. Neurones were acetoxymethyl ester preloaded with one of the mitochondrial calcium indicators Rhod-2, Rhod-FF or Rhod-5N (5μM for 30 min), and then whole cell patch clamped through a pipette containing the cytosolic calcium indicator Oregon Green 488 BAPTA-1 dextran (100μM OGBD, 10kD). Whole cell patch clamping allowed cytosolic Rhod dye to leave the cell at the same time as the cytosol loaded with OGBD. Depolarisation of the plasma membrane to +10mV for 1 s evoked a large Rhod-2 intensity increase in discrete organelles (N=11 cells). Colabelling with mitotracker green FM confirmed the identity of these organelles as mitochondria (N=7 cells). 50ms depolarizations evoked smaller but still significant signals from mitochondrial Rhod-2 (N=10 cells). When the lower affinity mitochondrial calcium indicator Rhod-FF (Kd = 19μM) was used, 1 s plasma membrane depolarisations evoked significant mitochondrial signals (N=5 cells), indicating that mitochondrial calcium elevations of micromolar amplitude are evoked by relatively brief episodes of calcium influx across the plasma membrane. To our surprise, inclusion of 100μM ruthenium red in the patch pipette did not significantly inhibit mitochondrial calcium uptake under these conditions (N=6 cells), suggesting that uptake occurs through a pathway distinct from the classical ruthenium red-sensitive calcium uniporter. These results suggest that mitochondrial calcium uptake will occur during normal electrical activity in sensory neurones and raise interesting questions about the mechanism by which the uptake occurs.