The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.