NRSF/REST is a protein that silences transcription of a number of genes that contain a DNA element called the neuron-restrictive silencer element (NRSE). During embryogenesis, REST is expressed ubiquitously in nonneural cells, but is down-regulated during differentiation of neural progenitors into neurons. REST is also up-regulated in adult neurons by activity, suggesting a possible role for the protein in synaptic plasticity. To understand mechanisms that control expression of REST, we identified and characterized the promoter region of the mouse REST gene (mREST). A 4.5-kb DNA segment containing three exons (A, B, and C) that correspond to alternatively spliced 5' untranslated regions (5'UTRs) was isolated and its DNA sequence was determined. Reverse transcription-PCR analyses of fibroblasts, astrocytes, and neural progenitors identified variants in which these exons were spliced to exon D, suggesting that exons A, B, and C may each have a promoter. Consistent with this hypothesis, primer extension and in vitro transcription experiments revealed clusters of RNA transcription initiation sites upstream of exons A, B, and C. Tests of REST/luciferase reporter constructs in Neuro2A and NIH 3T3 cells revealed promoters upstream of exons A and B that were active in both cell lines, and a promoter upstream of exon C that was weakly active only in NIH 3T3 cells. Six enhancer and two repressor regions were found to overlap each of the three promoters, and some of these were found to be cell type-specific. Combinatorial arrangements of these promoters with enhancer and repressor regions may allow modulation of REST expression in particular contexts.