Aim: The heart undergoes stages of postnatal development during which it responds differently to cardiac insults. Whether such age-related differences are due to developmental changes in single heart cells is not presently known. In this study we investigated the effect of chemical hypoxia on freshly isolated cardiomyocytes from different postnatal ages of the rat heart. Methods: Cardiomyocytes were enzymatically isolated from 14, 21 day and adult rat hearts. Isolated cardiomyocytes were perfused with HEPES buffer (1mM Ca²⁺) and field stimulated at 0.2Hz in a chamber under an inverted microscope at 34^οC. Chemical hypoxia was induced by switching the perfusion buffer to one containing 2.5mM NaCN and no glucose. Cardiomyocytes were viewed on a monitor and the effects of metabolic inhibition were noted. The times taken for the cells to cease contraction and to enter a state of rigor were recorded. Concentrations of ATP were measured in myocardial biopsies from the same aged rats, using HPLC. Data expressed as mean ± standard error. Results: The lengths of time for cardiomyocytes to both cease contraction and enter rigor were significantly shorter for cardiomyocytes from 21 day old rats compared to all other age groups (p<0.0001, ANOVA, n=30). Times taken to cease contraction were (in minutes) 12±2, 6±2, 17±3 for 14, 21 and adults respectively. This trend was mirrored in the times taken to enter rigor for all ages. Since going into rigor would depend on availability of myocardial ATP, we measured the concentration of ATP in myocardial biopsies from all age groups. Mean ATP levels in freshly excised hearts were 14.4±1.8, 12.5±1.6, 17.1±0.8 nmol/mg protein in 14, 21 days and adult respectively. ATP was significantly lower at 21 days compared to adults (p=0.024, unpaired t test, n=5). Conclusion: This work demonstrates that the age-related response to chemical hypoxia is likely to be due to differences in the basal levels of myocardial ATP.