Glutamate uptake by astrocytes is vital in the prevention of excitotoxicity, especially in circumstances such as stroke, when oxygen levels are severely compromised. However, the effects of hypoxia on glutamate transporter expression have not been studied in detail. Here, we report the effects of 24h hypoxia (1% O₂) on glutamate transporter levels in rat cortical astrocytes. Using subtype-specific antibodies, we identified the presence of transporters EAAT1, EAAT2 and EAAT3 in astrocyte homogenates (isolated as previously described; Smith et al., 2004) by western blotting. Exposure of astrocytes to hypoxia reduced protein EAAT1 levels by 39.4 ± 10.9% (n=5) and EAAT2 levels by 39.7 ± 6.0% (n=5). EAAT3 levels were unaffected. RT-PCR results show that the observed reduction in protein expression caused by hypoxia is mirrored by a reduction in mRNA levels of EAAT1 and EAAT2. The effects of hypoxia on protein expression levels of both EAAT1 and EAAT2 were prevented by the addition of the NF-kB inhibitor, SN50 (5μM), indicating a role for the transcription factor NF-κB in modulating the transcription and subsequent expression of EAAT1 and EAAT2. Immunocytochemistry also revealed that hypoxia caused translocation of NF-κB to the nucleus of the cell, and that this was prevented by SN50. NF-κB is known to activate, and be activated by, TNFα. As TNFα has been reported to regulate EAAT2 expression (Sitcheran et al., 2005) and as TNFα is produced by astrocytes following ischemic injury (Yu & Lau, 2000), we measured TNFα levels in astrocyte media following 24h hypoxia. Hypoxia caused a 7-fold increase in TNFα production, and application of exogenous TNFα (10ng/ml.) mimicked the effects of hypoxia on EAAT expression, indicating a possible role for TNFα in hypoxic regulation of the EAAT expression.