The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from -335 to -168 and -617 to -335, respectively. A strong His-1-positive regulatory element (HPRE, +18 to +29) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or SV40 promoters by greater than 80%. This negative regulatory sequence interacted with nuclear proteins from cells that contain a silent His-1 gene but showed no interaction with nuclear proteins from cells that actively transcribe the endogenous gene. HNRE-mediated repression was orientation independent; most of this activity was mapped to a minimal 26-bp sequence. These findings suggest that the first exon of the His-1 gene contains a cell type-specific silencer that contributes to the regulation of His-1 transcription.