While lifestyle and genetics are well-known risk factors for heart disease, the impact of the intrauterine environment on disease susceptibility is a relatively new concept. Insufficient oxygen supply to a foetus (prenatal hypoxia) has been linked with developmental programming and adverse effects in the adult heart of the offspring. Whole heart studies have suggested increased susceptibility to ischaemia reperfusion (I/R) injury but there are few studies that have investigated the underlying mechanisms. We set out to explore the effects of prenatal hypoxia on adult offspring cardiac mitochondria; we investigated mitochondrial oxygen consumption and reactive oxygen species (ROS) production. Pregnant C57 mice were subjected to 14% oxygen (hypoxic group) for gestational days 6-18. A control group (normoxic) were kept in 21% oxygen. Both groups delivered in 21% oxygen (normoxia) and the offspring were kept in normoxia for 7 months before being killed by cervical dislocation. Mitochondrial respiration and ROS production were assessed in homogenized left ventricular tissue using an Oroboros Oxygraph-O2k and O2k-Fluo LED2-Module. Data presented as mean ± SEM. Using a sequence of substrates and inhibitors, we found increased respiration and decreased ROS production in adult females previously subjected to prenatal hypoxia. The same protocol showed no significant differences in male respiration but increased ROS production in males from hypoxic pregnancies. The differences in female respiration were greatest during oxidative phosphorylation (517 ± 109 nmol O2-1sec-1mg in normoxic, n=7, compared to 957 ± 112 nmol O2-1sec-1mg in hypoxic offspring, n=8). Female ROS production showed the greatest difference during oxidative phosphorylation without activation of complex II (1.23 ± 0.25 pmol H2O2-1sec-1mg in normoxic, n=7 compared to 0.75 ± 0.12 pmol H2O2-1sec-1mg in hypoxic, n=8). Male ROS production also showed the greatest difference during oxidative phosphorylation without activation of complex II (0.43 pmol H2O2-1sec-1mg in normoxic,n=5, versus 0.93 pmol H2O2-1sec-1mg in hypoxic,n=8). In addition, enzyme activity assays showed that citrate synthase remained unchanged between the treatments, whilst complex I and II were decreased in hypoxic females, 73% and 62% respectively, compared to normoxic females. Complex IV activity increased with 92% in hypoxic females compared to normoxic females. No differences in enzyme activity were found in males. Our findings show that females respond differently to prenatal hypoxia than males at the mitochondrial level. The increase in basal ROS production in the hypoxic males could help to explain the increased susceptibility to I/R injury in males. In contrast, the lower basal ROS production in females from hypoxic pregnancies may protect the heart from I/R injury. In addition, our study further proves the need to explore physiology and pathology in females as well as males.