Hypoxia produces a greater dependence on glucose metabolism and anaerobic respiration for ATP production. Increases in glucose uptake can be mediated by insulin or IGF-I. The effect of combining these parameters on glucose uptake was studied in newborn canine ventricular heart cell cultures grown in DMEM with 10% calf serum. Tissue was obtained from fed pups within 12 hours of birth. Hypoxia (5% O2) and insulin or IGF-I increased basal 2-deoxyglucose uptake by 25-40% and the effects of low oxygen and hormones were additive. Insulin resistance did not exist in the heart cell cultures as it does in newborn animals. Western immunoblots showed that only GLUT-1 was present, unlike the donor tissue which had GLUT-1 and GLUT-4. A 24 hr treatment with low oxygen, 10 nM insulin or 10 nM IGF-I increased GLUT-1 protein by 47%, 22% and 96% respectively and did not cause expression of GLUT-4. Expression levels of insulin receptor subunits decreased by 0.5 fold in low oxygen but the decrease was inhibited by the presence of insulin or IGF-I. The hormones did not increase expression levels in normoxic cultures. Low oxygen (24 hr) had no effect on insulin binding but decreased IGF-I binding to high affinity sites by 50%. The data suggest: 1) that two pathways exist for glucose uptake regulation through GLUT-1 and 2) hypoxia perturbs the insulin and IGF-I action pathways.