We have identified a functional silencer element (positions -231 to -211) in the human synapsin I gene that selectively represses its transcription in nonneuronal cells. Transfection experiments using synapsin I-luciferase constructs show that site-specific mutations or deletion of this silencer sequence results in expression of the reporter gene in nonneuronal cells. Moreover, the silencer element is capable of conferring repression on a heterologous promoter in nonneuronal cells. Gel-shift assays reveal the presence of a sequence-specific synapsin I silencer-binding protein in nonneuronal cell extracts but not in neuronal cell extracts. Mutagenesis studies of the silencer sequence demonstrate that formation of the specific silencer-protein complex in vitro correlates well with repression of transcription in vivo. These data indicate that the interaction between synapsin I silencer and its binding protein is involved in tissue-specific expression of the synapsin I gene. In addition, our results suggest the existence of at least one additional cis-acting element within the promoter-proximal region (positions -233 to +20) that also contributes to the neuron-specific expression of the synapsin I gene.