We hypothesize that the primary mechanism for removal of glutamate from the extracellular space is altered after traumatic brain injury (TBI). To evaluate this hypothesis, we initiated TBI in adult male rats using a 2.0 atm lateral fluid percussion injury (LFPI) model. In the ipsilateral cortex and hippocampus, we found no differences in expression of the primary glutamate transporter in the brain (GLT-1) 24 h after TBI. In contrast, we found a decrease in glutamate uptake in the cortex, but not the hippocampus, 24 h after injury. Because glutamate uptake is potently regulated by protein kinases, we assessed global serine-threonine protein kinase activity using a kinome array platform. Twenty-five kinome array peptide substrates were differentially phoshorylated between LFPI and controls in the cortex, whereas 19 peptide substrates were differentially phosphorylated in the hippocampus (fold change >/= +/- 1.15). We identified several kinases as likely to be involved in acute TBI, including protein kinase B (Akt) and protein kinase C (PKC), which are well-characterized modulators of GLT-1. Exploratory studies using an inhibitor of Akt suggest selective activation of kinases in LFPI versus controls. Ingenuity pathway analyses of implicated kinases from our network model found apoptosis and cell death pathways as top functions in acute LFPI. Taken together, our data suggest diminished activity of glutamate transporters in the prefrontal cortex, with no changes in protein expression of the primary glutamate transporter GLT-1, and global alterations in signaling networks that include serine-threonine kinases that are known modulators of glutamate transport activity.