Stimulation of neurones with glutamate (Glu) induces cytosolic Ca²⁺ increases that can activate mitochondrial Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter. The present experiments investigated the effect of ageing on Glu-evoked cytosolic and mitochondrial Ca²⁺ signals. Primary cultures of cerebellar granule neurones (DIV 7–50) were loaded with Fura-2-AM (2.5mM) and stimulated with glutamate (2-200mM, in 0Mg/Gly) followed, after a wash period, by the protonophore CCCP (10mM) to unload the mitochondria. We show that with age in culture there is a significant increase in the amplitude of the Glu-evoked Ca²⁺ signals, associated with greater sensitivity of the neurones to Glu. The capacity of the mitochondria to accumulate Ca²⁺ was not affected by age and the larger cytosolic Ca²⁺ increases found in the older neurones led to larger mitochondrial Ca²⁺ accumulation. The correlation between the Glu-evoked signals and the subsequent CCCP-releasable Ca²⁺ responses was similar in the young and old neurones. Despite the fact that in the aged neurones, including those in cultures, the mitochondria are chronically depolarised, our data suggest that within the normal range of physiological responses, the key factor which determines the mitochondrial Ca²⁺ load is the size of the cytosolic Ca²⁺ signal.