Notch signaling is involved in many cell fate determination events in metazoans. Ligand binding results in proteolytic cleavage to release the signal-transducing Notch intracellular domain (NICD). The nuclear protein RBP-J kappa, when complexed with NICD, acts as a transcriptional activator which, in turn, induces a target gene of Notch such as the repressors HES/E(spl) and HERP2. Under physiological stimulation using co-culture with Notch ligand-expressing cells and target cells expressing Notch receptors, the HES1 gene and the HERP2 gene have been shown to be directly up-regulated by Notch ligand binding. However, expression of another member of the HERP family, HERP1, was not induced by ligand stimulation in any cells tested, leading to the suggestion that HERP1 may not be an immediate target of Notch or that Notch pathways can be cell type-specific. Because HERP1 appears to play a central role in the development of the aorta (Zhong, T. P., Rosenberg, M., Mohideen, M. A., Weinstein, B., and Fishman, M. C. (2000) Science 287, 1820-1824), we re-addressed the issue of its relationship with the Notch pathway by examining its expression in A10 smooth muscle cells derived from thoracic aorta. We show that in these specific cells HERP1 is also a direct target gene of Notch. NICD activates the HERP1 promoter in an RBP-J kappa-dependent manner, and induces expression of endogenous HERP1 mRNA as well as HERP1 protein in A10 cells. Co-culture with Notch ligand-bearing cells induces endogenous HERP1 mRNA expression in A10 cells, and these events occur even in the absence of de novo protein synthesis. In addition, RBP-J kappa proved essential for induction of HERP1 mRNA in Notch signaling because exogenous RBP-J kappa was sufficient to rescue HERP1 mRNA expression in RBP-J kappa-deficient cells. These findings provide the first solid evidence that HERP1 is a novel primary target of Notch and underscores the cell-specific complexity of the Notch regulatory pathway. Given that Notch signaling plays a crucial role in vascular development, Notch may derive its function via HERP family members.