Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor which is both present on the cell surface and able to generate an intracellular signal in response to ligand. Consistent with previous reports, we found that disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbed ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL (CBF1, Su(H), and LAG-1). Yet contrary to these reports, we could detect the full-length p300 Notch1 product on the cell surface. Moreover, this uncleaved form of Notch1 could suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support our previous studies characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. Our results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor could mediate two distinct signaling pathways in response to ligand.