The interactions between Notch (N) receptors and their transmembrane ligands, Jagged1 (JI) and Delta1 (Dl1) mediate signaling events between neighboring cells that are crucial during embryonal development and in adults. Since the non-transmembrane extracellular form of J1 acts as an antagonist of N activation in NIH 3T3 mouse fibroblast cells and induces an FGF1-dependent transformed phenotype (Small, D., et al. (2003) Notch Activation Suppresses FGF-dependent Cellular Transformation, J. Biol. Chem., 278:16405-13), we examined the potential redundant functions of the two subfamilies of Notch ligands and report that while the soluble (s) forms of both Dl1 and J1 act as N signaling antagonists in NIH 3T3 cells, they do display disparate functions. Interestingly, while sJ1 induced an attenuation of cell motility which is accompanied by a decrease in actin stress fibers and focal adhesion sites and increase in adherence junctions, sDl1 does not. In contrast, however, sJ1, like sDl1, induces a NIH 3T3 cell tranformed phenotype mediated by FGF receptor (R) signaling. Lastly, because the inhibition of classical N signaling by sJ1 and sDl1 is rescued by dominant-negative Src expression, we suggest that there may be cooperation between the classical Notch signaling and the Src signaling pathways.