Intramitochondrial free calcium ([Ca²⁺]_m) is an important regulator of ATP production in the heart, and may also modulate whole-cell Ca²⁺ signalling. However, research has been hampered by the difficulty of measuring [Ca²⁺]_m in living myocytes. Here, we used an adenovirus containing aequorin targeted to either cytosol or mitochondria to determine whether beat-to-beat oscillations in [Ca²⁺]_m could be detected in adult myocytes. Adult rats were humanely killed and ventricular myocytes isolated by collagenase digestion. Myocytes were then cultured in the presence of adenovirus containing aequorin targeted to either mitochondria or cytosol. [Ca²⁺] was measured in cells electrically stimulated at 2Hz by collecting aequorin light output using a photon counting camera (10-50 cells per field). Results are presented as means ± S.E.M.; n values refer to the number of separate fields of cells studied. Correct localisation of the aequorin constructs to the mitochondria and cytosol was confirmed by immunocytochemistry using antibodies against the Haemagglutinin tag contained in the constructs. The images obtained were indicative of correct compartmentalisation of the expressed proteins. Ca²⁺ transients were clearly visible in both cytosol and mitochondria. Although resting (diastolic) values could not be accurately estimated due to the high signal to noise ratio at low [Ca²⁺], systolic values were quantifiable. In the presence of the β adrenergic agonist isoproterenol systolic [Ca²⁺]_c increased from 0.9 μM ± 0.04 (n=36) to 1.65 μM ± 0.07 (n=19) and [Ca²⁺]_m from 0.9 μM ± 0.09 (n=11) to 1.2 μM ± 0.11 (n=10). We also performed these experiments in the presence of the mitochondrial Na⁺-Ca²⁺ exchange inhibitor clonazepam. Addition of 50 μM clonazepam caused a transition from beat-to-beat transients to a sustained [Ca²⁺] of 1.2 μM ± 0.08 (n=16) in mitochondria (in the presence of isoproterenol). Conversely, clonazepam reduced the amplitude of cytosolic transients from 1.0 μM ± 0.02 (n=10) to 0.79 μM ± 0.04 (n=10) and from 1.91 μM ± 0.09 (n=8) to 1.48 μM ± 0.09 (n=10) in the presence of isoproterenol. Since changes in [Ca²⁺]_m are known to regulate mitochondrial energy production, mitochondrial [ATP] was measured using the expression of targeted luciferase. No beat-to-beat changes in [ATP]_m could be observed using this method, although a sustained rise in [ATP]_m was observed over longer time periods when myocytes were stimulated from rest in the presence of isoproterenol. These results implicate the mitochondrial efflux pathway in modulation of cytosolic Ca²⁺ transients. The lack of change in mitochondrial ATP in beating myocytes indicates that ATP supply and demand are normally well matched. However, [ATP]_m can increase upon rapid stimulation of resting cells.