Postnatally acquired dietary iron deficiency has adverse long-term effects on neurodevelopment. Recently, our laboratory has identified gestational conditions (diabetes mellitus, intrauterine growth retardation) which result in a significant prevalence of perinatal brain iron deficiency. These groups of infants have poorer long-term developmental outcome, but the role of iron deficiency is unknown. Loss of cytochrome oxidase (CytOx) activity in the region of the hippocampus, an area central to cognitive function (recognition memory), has been shown to be an early and sensitive indicator of neuronal damage following perinatal hypoxic-ischemic encephalopathy (HIE) in the rat. We hypothesized that perinatal iron deficiency enhances the vulnerability of the hippocampus to HIE injury. Twenty 7-day-old, iron-deficient rat pups and 20 iron-sufficient controls underwent right carotid artery ligation and were exposed to 2.5 hours of hypoxia (8% FIO2). In this model (the Levine model), both hypoxia and ischemia (right side), but not hypoxia alone (left side), are needed to induce changes in neuronal CytOx activity. The left side is thus the internal control. The mean ± SD brain iron concentration of the iron-deficient group was 40% lower than the iron-sufficient group (28.0± 6.8 v 51.0 ± 7.8; p<0.001). The CytOx activity, visualized with diaminobenzedine, in four different areas of hippocampus 24 hours after hypoxic-ischemic damage was measured in optical density (OD) units (range: 1 black/254 white) by computer-assisted densitometry. There was right-sided CytOx activity loss in the dentate gyrus (102 ± 16 v 89 ± 17 OD units; p=0.009), CA3c (105 ± 16 v 93 ± 19 OD units; p=0.003) and CA1 (108 ± 27 v 98 ± 17 OD units; p=0.06) compared to the unligated left side in the iron-deficient group. Side-to-to-side differences were seen only in CA1 in the iron-sufficient group. Overall, the iron-deficient group had more subareas with right-sided loss than the iron-sufficient group (p=0.04), and the magnitude of right-left differences was greater in the iron-deficient group (p=0.05). Perinatal iron deficiency enhances the vulnerability of the neonatal brain to HIE and may contribute to long-term neurodevelopmental delays in at-risk newborns. Supported by NICHD.