This study tested the hypothesis that specific hypoxic molecules, including HIF-1, nNOS and VEGF, are up-regulated within the cerebral cortex of acutely anemic rats. Isoflurane anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial P_aO₂ values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 ± 2 g^.L^-1. In anemic rats, cerebral cortical HIF-1 protein levels were increased, relative to controls (1.7 ±0.5 fold, p0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS and nNOS (p0.05 for all), but not eNOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 ± 0.2 and 1.5 ± 0.4 fold respectively, p0.05 for both). Immunohistochemistry demonstrated increased HIF-1 and VEGF staining in peri-vascular regions of the anemic cerebral cortex and an increase in the number of nNOS positive cerebral cortical cells (3.2 ± 1.0 fold increase, p<0.001). Immunofluorescence demonstrated that nNOS positive cells co-stained with the neuronal marker, Neu-N, but not with the astrocytic marker GFAP. The nNOS positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining co-localized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1 protein and mRNA for HIF-1 responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.