AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors are assembled from four subunits, GluR1-4. Although GluR4 is widely expressed in brain its abundance is less than GluR1-3. We have isolated approximately 5 kb of the rat GluR4 promoter region and analyzed its capacity to drive expression of a luciferase reporter gene in transfected rat cortical neurons and glia, and C6 glioma cells. Multiple transcriptional start sites were identified in a GC-rich region lacking TATA-boxes between -1090 and -1011 bp from ATG. In transfected mixed cortical cultures, luciferase expression driven by GluR4 promoter segments were found predominantly in TuJ1-positive neurons, indicating neuronal preference of GluR4. The GluR4 promoter fragments were 6-12-fold more active in neurons than glia, compared with a 30-fold neuronal selectivity of GluR2. Deletion of the GluR4 transcriptional initiation region decreased luciferase activity in neurons, but increased activity in C6 cells, suggesting that regulatory elements governing neuronal expression reside in this region. An intron within the 5'-untranslated region and Sp1, IK2 and E-box sites are conserved in the rat, mouse and human GluR4 promoters. The relative activity of GluR4 and GluR2 promoters in transfected cells correlates with their expression in brain, and in both promoters regulatory elements for neuronal expression reside near the initiation sites.