Energy metabolism changes dramatically during post-natal development of the heart, e.g., a shift from carbohydrates to fatty acids as the major fuel for ATP production (Marin-Garcia et al., 1997). In adult myocytes, mitochondrial respiration is stimulated by an increase in mitochondrial free calcium ([Ca²⁺]_m) (McCormack et al., 1990), which allows the heart to increase its ATP supply rapidly with changes in physiological conditions. However, it is not known whether this regulation is present in neonatal hearts. Our aims were to determine whether basal respiration rates altered during development from neonate to adult, and to establish whether Ca²⁺ increased respiration in neonatal as well as adult hearts. All rats were killed humanely by cervical dislocation. Mitochondria were isolated from hearts of neonatal (2-3 day old), and adult (2-3 month old) rats by differential centrifugation, including a percoll purification step to remove contaminating membrane fragments. Absence of plasma membrane in the final preparation was confirmed using an antibody to rat monocarboxylate transporter-1. Results are expressed as means ± S.E.M. and unpaired t-test used for statistical analyses. Mitochondrial content of the hearts increased by approximately 60% in the adult compared with neonatal hearts, as determined from the activity of citrate synthase, a mitochondrial marker enzyme. Maximal rates of ADP-stimulated respiration using saturating concentrations of the substrates glutamate and malate were 100 ± 16 nmoles O/min/mg protein (n=11) in neonates and 284 ± 24 nmoles O/min/mg protein (n=27) in adults. The efficiency of coupling of respiration to ATP synthesis (ratio of rates + ADP: -ADP) was also higher in adult hearts (not shown). To determine an effect of Ca²⁺ on respiration, oxidation of 2oxoglutarate was used at low rate limiting concentrations, 0.5 mM, since oxoglutarate dehydrogenase is known to be Ca²⁺-sensitive in adult hearts (McCormack et al., 1990). In mitochondria from adult hearts, addition of 35 nM Ca²⁺ resulted in an increase in respiration by 50% (n=7; p<0.05), whereas respiration in neonatal hearts (n=3) was unchanged. We have developed methods for isolating pure mitochondrial fractions from rat hearts at different stages of development. ADP-stimulated respiration rates were higher in adult compared with neonatal hearts, as was the degree of coupling of respiration to ATP synthesis. The ability of Ca²⁺ to stimulate respiration appears to be acquired during development from neonate to adult. Further work is needed to determine the exact stage at which this occurs, and the possible mechanisms involved.