The hippocampus is injured in both hypoxia-ischemia (HI) and perinatal iron deficiency that are co-morbidities in infants of diabetic mothers and intrauterine growth restricted infants. We hypothesized that preexisting perinatal iron deficiency predisposes the hippocampus to greater injury when exposed to a relatively mild HI injury. Iron-sufficient and iron-deficient rats (hematocrit 40% lower and brain iron concentration 55% lower) were subjected to unilateral HI injury of 15, 30, or 45 mins (n=12 to 13/HI duration) on postnatal day 14. Sixteen metabolite concentrations were measured from an 11 μL volume on the ipsilateral (HI) and contralateral (control) hippocampi 1 week later using in vivo 1H NMR spectroscopy. The concentrations of creatine, glutamate, myo-inositol, and N-acetylaspartate were lower on the control side in the iron-deficient group (P<0.02, each). Magnetic resonance imaging showed hippocampal injury in the majority of the iron-deficient rats (58% versus 11%, P<0.0001) with worsening severity with increasing durations of HI (P=0.0001). Glucose, glutamate, N-acetylaspartate, and taurine concentrations were decreased and glutamine, lactate and myo-inositol concentrations, and glutamine/glutamate ratio were increased on the HI side in the iron-deficient group (P<0.01, each), mainly in the 30 and 45 mins HI subgroups (P<0.02, each). These neurochemical changes likely reflect the histochemically detected neuronal injury and reactive astrocytosis in the iron-deficient group and suggest that perinatal iron deficiency predisposes the hippocampus to greater injury from exposure to a relatively mild HI insult. © 2007 ISCBFM All rights reserved.