The importance of mitochondrial free calcium ([Ca²⁺]_m) in whole-cell Ca²⁺ signalling has been shown in several non-cardiac cell types. However, whether [Ca²⁺]_m plays a role in beat-to-beat Ca²⁺ regulation in the heart is controversial, due to the difficulty of measuring [Ca²⁺]_m in living myocytes. Recently Robert et al. demonstrated the existence of [Ca²⁺]_m transients in neonatal rat myocytes using the Ca²⁺ sensitive photoprotein aequorin targeted specifically to mitochondria. Here, we use adenoviruses to target aequorin to either the cytosol or mitochondria of neonatal and adult myocytes in order to determine whether beat to beat oscillations in [Ca²⁺]_m occur in both systems.Adult and neonatal rats were humanely killed by cervical dislocation and ventricular myocytes isolated by digestion using collagenase (adult) or trypsin (neonates). Myocytes were cultured for 24-72 hours in the presence of adenovirus containing aequorin targeted to either mitochondria or cytosol ².All expermiments were performed using 2 mM extracellular [Ca²⁺]. [Ca²⁺] was measured in either the spontaneously beating neonatal myocytes, or electrically stimulated (2Hz) adult myocytes by collecting aequorin light output using a photon counting camera. Light was collected from a field of approximately 50 cells, and results are presented as means ± S.E.M.; n values refer to the number of separate fields of cells studied. In neonatal myocytes, our results agreed broadly with those of Robert et al., and we were able to detect clear Ca²⁺ transients in both cytosol and mitochondria. In adult cells, cytosolic Ca²⁺ transients were clearly visible, although resting (diastolic) values were somewhat variable due to the low sensitivity of aequorin at diastolic [Ca²⁺]. At peak systolic [Ca²⁺] aequorin was more sensitive, allowing calibration of the signal. This gave a systolic value of 1.0 ± 0.05 µM (n=6), increasing to 1.4 ± 0.08 µM (n=6) in presence of 10 µM isoproterenol. In mitochondria, no changes in [Ca²⁺]_m could be detected beat-to-beat unless isoproterenol was added, when [Ca²⁺]_m transients were clearly visible, with a peak value of 1.31 ± 0.09 µM (n=5). In conclusion, we have successfully transfected both adult and neonatal myocytes with aequorin targeted to either cytosol or mitochondria. Although beat-to-beat Ca²⁺ transients were detected in mitochondria of neonatal cells, this ability seems to be lost during development to adulthood, unless the cells are additionally stimulated with the β-adrenergic agonist isoproterenol. Further work is needed to determine the implications of these findings to physiological and pathological Ca²⁺ signalling in the heart