Repressor Element-1 Silencing Transcription factor (REST) is a negative regulator of neuronal genes that contain a 23 bp repressor element (RE1) (Schoenherr & Anderson, 1995). Although REST has been associated almost exclusively with neuronal phenotype we provide evidence for a role for REST in regulating the expression of KCNN4 in vascular smooth muscle cells. Male 8-week-old mice were humanely killed. Using a rabbit polyclonal antibody targeted to REST we detected a 115kDa protein band in mouse aorta. This is close to the predicted molecular mass of full-length REST (117kDa). Using RT-PCR we also detected REST mRNA in isolated smooth muscle cells from mouse aorta. To link REST expression to function, we used a bioinformatics search and identify conserved RE1 sites within the promoter region of human, mouse and rat KCNN4 gene, which encodes the intermediate calcium-activated potassium channel IKCa. To determine whether REST has a functional role in regulating the KCNN4 promoter, we cloned a region of the KCNN4 gene into a luciferase reporter plasmid. When transfected in REST-expressing HEK cells, the promoter containing a mutation of the RE1 site drove a 27-fold higher luciferase expression than the wild type promoter. Gel electromobility shift assays show that the RE1 site of the KCNN4 formed a DNA-protein complex and that vascular smooth muscle nuclear extracts contain REST protein bound to this RE1 site. Furthermore introduction of a dominant-negative form of REST to isolated aorta smooth muscle cells in primary culture induces the upregulation of KCNN4 gene expression. Together, these data indicate a novel role for REST in regulating KCNN4 gene expression in vascular smooth muscle cells.