High-affinity glutamate uptake (HAGU) transporters rapidly remove released glutamate from the synaptic cleft. If HAGU is suppressed, neurotoxic concentrations of excitatory amino acids may accumulate. To seek further evidence in support of the neurotoxicity of endogenous glutamate in the developing brain, we assessed the neurotoxicity of the selective HAGU inhibitor L-trans-2,4-pyrrolidine dicarboxylate (L-PDC) in postnatal day 7 (PND 7) rats. The hippocampus of PND 7 rats is susceptible to EAA agonist-mediated injury; features of injury include atrophy and neuronal loss. Since HAGU is energy-dependent, we hypothesized that moderate hypoxia would increase L-PDC-mediated injury by further suppressing HAGU. L-PDC was stereotaxically injected into dorsolateral hippocampus of PND 7 rats (568 nmol, n = 20). Prior to return to the dam, rats were divided into two groups, one of which was subjected to moderate hypoxia (3 h, FiO2 = 0.08) (n = 11; 2 died acutely). On PND 12, hippocampal neuropathology was assessed by a blinded observer using a five-point scale and also by measuring hippocampal cross-sectional areas with computerized image analysis. Three brains were excluded from analysis, since markedly asymmetric tissue sectioning precluded valid side-to-side comparison of hippocampal areas. Injection of L-PDC alone elicited focal pyramidal cell loss (6/7); in the (L-PDC + hypoxia) group, injury was significantly increased (median scores: L-PDC = 2; [L-PDC + hypoxia] = 3.5; p < 0.005). Hippocampal atrophy was noted only after L-PDC + hypoxia (4/8) (percent right-left difference in mean hippocampal area [+/- SE]: L-PDC = 2.5% [+/- 2.6]; [L-PDC + hypoxia] = 8.9% [+/- 3.2]; p < 0.02). In tissue from PND 7 rats, L-PDC (10 microM) inhibited hippocampal synaptosomal HAGU by > 85%; at the same concentration, L-PDC did not displace [3H]glutamate from NMDA- or AMPA-sensitive hippocampal binding sites. These results support the hypothesis that increased synaptic accumulation of endogenous excitatory amino acid neurotransmitters may produce hippocampal injury in perinatal rodents.