The Kv3.1 potassium channel is expressed in neurons that generate trains of high frequency action potentials in response to synaptic inputs. To understand the mechanisms underlying the regulation and restricted expression pattern of the Kv3.1 gene, we have cloned and characterized its promoter. We first isolated a 5.3-kilobase pair fragment of the Kv3.1 5'-flanking region. When linked to the chloramphenicol acetyltransferase reporter gene, this fragment was found to be active in the undifferentiated PC12 cell line, a neuron-like cell line, but not in a fibroblast cell line. By carrying out a series of deletion analyses in undifferentiated PC12 cells, we have localized the essential promoter region to a highly GC-rich region containing four Sp-1 binding sites. Similar deletion analysis in NIH3T3 cells suggests that multiple silencing elements and enhancing element(s) are involved in the cell type-specific expression of this gene. Further regulatory elements, including one cyclic AMP/calcium response element (CRE) and one Ap-1 element were found in the upstream region of the promoter. Using a stable undifferentiated PC12 cell line transfected with the Kv3.1 5'-flanking region, we determined that promoter activity is enhanced by a cAMP analog and a calcium ionophore. Deletion of the CRE-like element at position -252 eliminated the enhancement of promoter activity by cAMP, and mobility shift assays confirmed that the Kv3.1 CRE sequence binds both a nuclear factor in undifferentiated PC12 cells and recombinant CRE binding protein. Our results suggest that the transcription of the Kv3.1 channel may be regulated by neurotransmitters that elevate cAMP levels in neurons.