Myocyte physiology changes dramatically during development from neonate to adult, e.g., changes in cell morphology, Ca²⁺ signalling, and energy metabolism (Marin-Garcia et al., 1997). This may be responsible for the differing response of neonatal and adult hearts to ischaemia/reperfusion injury, in particular the role played by mitochondria. Our aims were to isolate cardiomyocytes from rats of different ages (from neonate to adult), and to develop conditions for using fluorescent indicators of mitochondrial function: mitochondrial membrane potential (∆ψ _m) and mitochondrial free calcium ([Ca²⁺]_m).Rats were killed humanely by cervical dislocation. Ventricular myocytes were isolated from neonatal (2 day old) rats and adult (2-3 month old) rats by standard methods. To isolate myocytes from immature rats (7, 14 and 21 day old), a constant pressure system of perfusion had to be employed. Although we were able to isolate viable cells using this method, the cells appeared to be very fragile and did not survive loading with fluorescent indicators. Therefore only neonatal and adult cells were used in subsequent studies. Several dyes can be used to measure ∆ψ ,but only rhodamine 123 gave consistent results in our experiments. This dye accumulates in mitochondria of healthy cells because of their high ∆ψ _m.Exposure of cells to the uncoupler FCCP caused mitochondria to depolarise, with loss of the indicator. Fluorescence then increased by about 50% in both adult and neonatal cells due to dequenching of the dye in the cytosol. We have previously used indo-1 to measure [Ca²⁺]_m in adult myocytes, but this dye did not distribute well into mitochondria of neonatal cells. However, the Ca²⁺ indicator rhod2 did localise to mitochondria, but only under very specific loading conditions: cells were incubated for 2 hours at 4^oC, followed by incubation at 37^oC for 30 min. The distribution pattern of rhod2 was identical to that of rhodamine 123. In summary, we have developed conditions for detection of both ∆ψ _m and [Ca²⁺]_m in single living myocytes from neonatal and adult rats. Much of the literature regarding a role of [Ca²⁺]_m in both physiological and pathological cell Ca²⁺ signalling in the heart is controversial, at least partly because different groups have used different indicators, and cells from different species or state of development. To the best of our knowledge, this is the first time that these dyes have been successfully loaded into mitochondria in cells from both neonates and adults in the same study. This will allow direct comparison of the role of mitochondria in neonates and adults, both in normal cell Ca²⁺ signalling and in models of diseases such as ischaemia/reperfusion injury.