The mechanisms of specification of gene expression in a complex tissue such as the brain remain poorly understood. To provide a model system for the study of gene regulation in a specific subpopulation of differentiated neurons, we have derived cell lines from tumors created in transgenic mice by targeting simian virus 40 T antigen expression by using the regulatory regions of the gene for gonadotropin-releasing hormone (GnRH), a decapeptide released from specialized neurons in the hypothalamus. Transfections into the cultured GnRH-secreting hypothalamic neuronal cell line GT1 have identified a neuron-specific enhancer, 1.5 kb upstream of the GnRH gene, which binds multiple GT1 nuclear proteins. In particular, one AT-rich protein-binding region, AT-a, is critical for enhancer activity. In this study, we used electrophoretic mobility shift assays to detect a GT1 nuclear protein complex that binds the AT-a region. Close inspection of the AT-a bottom-strand sequence revealed homology to the octamer motif, a sequence known to bind members of the POU homeodomain transcription factor family. Although we demonstrate expression of a number of POU homeodomain genes in GT1 cells, a supershift assay with Oct-1 antibody demonstrates that Oct-1 is the protein binding the enhancer. Finally, specific mutations in the AT-a region that affected Oct-1 binding were correlated with decreased transcription. Thus, Oct-1 binds to the GnRH enhancer in vitro, and this binding is critical to the transcriptional activity of this neuron-specific enhancer in GT1 cells.