Phosphorylation of Notch proteins has been indirectly correlated with Notch activation and nuclear translocation as well as cellular transformation. There is evidence that the Wnt signaling pathway, which results in glycogen synthase kinase-3 beta (GSK-3 beta) inhibition, cross-talks with the Notch pathway. In this study, we show that GSK-3 beta is able to bind and phosphorylate Notch2 in vitro and in vivo. We identify three specific phosphorylation sites in the Notch2 serine/threonine-rich domain that are dependent on GSK-3 beta activity. Phosphorylation of the serine/threonine-rich domain has been shown previously to be crucial in regulating cytokine-specific cell differentiation. Coimmunoprecipitation experiments show that full-length Notch2 binds more efficiently than intracellular Notch2 to GSK-3 beta. Nevertheless, only the processed Notch2 is a substrate for the kinase, thus suggesting that GSK-3 beta-dependent phosphorylation may be specifically regulating the activated Notch molecule. Consistent with this, GSK-3 beta inhibits the transcriptional activation of Notch target genes both in vitro and in vivo, whereas lithium chloride treatment or Wnt-1 overexpression that results in GSK-3 beta inhibition leads to the up-regulation of the Hes-1 promoter. Together, our results suggest that cross-talk between Notch and Wnt pathways may be partially mediated by specific regulation of GSK-3 beta-dependent Notch phosphorylation.