The Kv3.1 potassium channel gene is restrictively expressed in the CNS, and its expression level is especially high in neurons that are able to follow synaptic inputs at high frequencies. To understand the transcriptional mechanisms controlling Kv3.1 expression, we have conducted a functional analysis of the Kv3.1 promoter in various cell lines of different tissue origins and in transgenic mice. Our results suggest that an upstream regulatory fragment coupled with the 5' untranslated region (UTR) is able to confer tissue-specific expression in both cell lines and in transgenic mice. Deletion analysis of the regulatory region carried out in cell lines reveals that a strong negatively acting element, uniquely residing in the 5' UTR (+350 to +158), appears able to confer cell type specificity on both the Kv3.1 promoter and the thymidine kinase promoter in transient transfection assays. A weak cell type-specific enhancer in the proximal region of the promoter (-123 to -71) also contributes to cell type-specific expression of the Kv3.1 gene.