Abstract—A novel approach with chimeric SM22&agr;/telokin promoters was used to identify gene regulatory modules that are required for regulating the expression of genes in distinct smooth muscle tissues. Conventional deletion or mutation analysis of promoters does not readily distinguish regulatory elements that are required for basal gene expression from those required for expression in specific smooth muscle tissues. In the present study, the mouse telokin gene was isolated, and a 370-bp (−190 to 180) minimal promoter was identified that directs visceral smooth muscle–specific expression in vivo in transgenic mice. The visceral smooth muscle–specific expression of the telokin promoter transgene is in marked contrast to the reported arterial smooth muscle–specific expression of a 536-bp minimal SM22&agr; (−475 to 61) promoter transgene. To begin to identify regulatory elements that are responsible for the distinct tissue-specific expression of these promoters, a chimeric promoter in which a 172-bp SM22&agr; gene fragment (−288 to −116) was fused to the minimal telokin promoter was generated and characterized. The −288 to −116 SM22&agr; gene fragment significantly increased telokin promoter activity in vascular smooth muscle cells in vitro and in vivo. Conversely, a fragment of the telokin promoter (−94 to −49) increased the activity of the SM22&agr; promoter in visceral smooth muscle cells of the bladder. Together, these data demonstrate that both vascular- and visceral smooth muscle–specific regulatory modules direct gene expression in subsets of smooth muscle tissues.