We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), an antagonist of N-methyl-d-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [³H]MK-801 binding in the hippocampus and [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [³H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.