Recent chick experiments have shown that Notch signaling plays context-dependent distinct roles in inner ear development: initially, Notch activity confers a prosensory character on groups of cells by “lateral induction”; subsequently, it is involved in the establishment of fine-graded patterns of hair cells and supporting cells by “lateral inhibition.” However, the spatiotemporal pattern of Notch activation in situ during mammalian inner ear development has not been investigated. In this study, we detected the expression patterns of the activated form of Notch1 (actN1) as well as those of endogenous Notch1, Jagged1 (Jag1), and Math1. ActN1 was detected by immunohistochemistry using an antibody that specifically recognizes the processed form of the intracellular domain of Notch1 cleaved by presenilin/γ-secretase activity. Between embryonic days (E)12.5 and E14.5, actN1 was weakly detected mainly in the medial region of cochlear epithelium, where Jag1-immunoreactivivty (IR) was also observed. Jag1-IR gradually became stronger in a more sharply defined area, finally becoming localized in supporting cells, while actN1 was detected in an overlapping area. Thus, a positive feedback loop was assumed to exist between the expression of Jag1 and actN1. In addition, actN1 started to be strongly expressed in the cells surrounding Math1-positive hair cell progenitors between E14.5 and E15.5. Strong actN1-IR continued in both a supporting cell lineage and in the greater epithelial ridge during the perinatal stage but ended by P7, suggesting that Notch1 activation may initially demarcate a prosensory region in the cochlear epithelium and then inhibit progenitor cells from becoming hair cells via classical “lateral inhibition.” J. Comp. Neurol. 497:502–518, 2006. © 2006 Wiley-Liss, Inc.