In adult myocytes, mitochondrial respiration is stimulated by an increase in intramitochondrial free calcium ([Ca²⁺]_m), which allows the heart to increase its ATP supply rapidly with changes in physiological conditions. However, it is not known whether this ability is present from birth, or acquired during development. The aims of this study were to establish whether Ca²⁺ increased respiration rates and 2-oxoglutarate dehydrogenase (OGDH) activity in rat heart mitochondria during postnatal development. Rats were humanely killed, and mitochondria isolated from hearts of neonatal (2-3 day old), 7, 14, 21 day old, and adult (2-3 month old) male Wistar animals. The rate of respiration was measured by following oxygen consumption of intact mitochondria using an oxygen electrode, and OGDH activity in permeabilised mitochondria by monitoring production of NADH using a spectrophotometric assay. Results are expressed as means ± S.E.M. and statistical significance determined by 1-way ANOVA. n = at least 6 separate preparations of mitochondria. Using 0.5 mM [2-oxoglutarate], which is rate-limiting in mitochondria from adult rats, 0.6 μM [Ca²⁺] stimulated respiration approximately 2-fold (p<0.05) in mitochondria from adult and 21-day-old rat hearts; but this effect was not observed in the 2, 7, or 14 day olds. However, at 0.1 mM [2-oxoglutarate], 0.6 μM [Ca²⁺] stimulated respiration in both adult and neonatal mitochondria (p<0.01). Thus at 0.5 mM 2-oxoglutarate in the neonates, high rates of respiration were observed even in the absence of [Ca²⁺]. When OGDH activity was measured, 0.6 μM Ca²⁺ increased enzyme activity in all age groups when either 0.1 or 0.5 mM [2-oxoglutarate] was used. However, kinetic measurements indicated that affinities of the enzyme for its substrate differed: K_m was 0.92 ± 0.15 vs. 0.52 ± 0.06 mM (p<0.05), and V_max 47.7 ± 5.7 vs. 18.6 ± 3.3 nmol min^-1 mg^-1 in adults vs. neonates, respectively. 1 μM Ca²⁺ reduced the K_m of the enzyme from 0.92 ± 0.15 to 0.17 ± 0.01 mM (p<0.05) in the adult and 0.52 ± 0.06 to 0.04 ± 0.02 mM (p<0.05) in the neonate, without affecting V_max. In conclusion, Ca²⁺ can stimulate respiration at all stages of development, which correlates with activation of OGDH by Ca²⁺. This suggests the presence of Ca²⁺-sensitive respiration from birth; however, the higher affinity of OGDH for its substrate may overcome any effect of Ca²⁺ in mitochondria from neonatal rat hearts.