Abstract: Dopamine β-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons in the nervous system. Transient transfection assays have indicated that cell-specific transcription of the human DBH gene may require a cell-specific silencer region residing at −486 to −263 bp upstream of the transcription start site. This region includes a putative DBH negative regulatory element (DNRE) with sequence homology to the restrictive element-1 (RE1)/neuron-restrictive silencer element identified in many other neural-specific genes. However, DNRE exerted negative regulation in both neuronal and nonneuronal cells alike, and site-directed mutation of this element did not significantly diminish the repressive activity of the DBH silencer region. Furthermore, expression of RE1-silencing transcription factor/neuron-restrictive silencer factor repressed neither DBH nor tyrosine hydroxylase promoter activity. We now report identification of three protein binding sites in the silencer region of the human DBH gene: SI at −271 to −250 bp, SII at −316 to −295 bp, and SIII at −348 to −324 bp. In vitro binding studies showed that SI and SIII, but not SII, interact with nuclear proteins from DBH-negative cells in a cell-specific manner. Furthermore, SI and SIII preferentially repressed the heterologous thymidine kinase and homologous DBH proximal promoter activities in nonneuronal cells. Taken together, the cell-specific silencer function of the upstream DBH region appears to involve several cis-regulatory elements, including two cell-specific repressor elements, SI and SIII, and a general negative regulatory element, DNRE. Based on these data, we propose that a highly restricted pattern of DBH gene expression in (nor)adrenergic cells of the nervous system may be controlled by multiple negative regulatory elements/silencers.