Although the intracellular domain of Notch1 is phosphorylated and it associates with members of the CSL family, the relationship of these events is poorly understood. Using in vivo [P-32]orthophosphate labeling of cells expressing transfected Notch1, we observed that the furin cleaved Notch1 (TMIC) and the soluble intracellular forms (NICD), but not the full-length molecule were phosphorylated. Furthermore, transfected NICD molecules showed a significantly greater specific activity of phosphorylation, or hyperphosphorylation, compared to TMIC molecules. Hyperphosphorylation of NICD was also observed when NICD was generated by an endogenous intramembraneous cleavage of TMIC. However, TMIC molecules bearing a mutation that reduces intramembraneous cleavage (V1744K) did not show an enhanced incorporation of phosphate, suggesting that cleavage is required for hyperphosphorylation. Using deletion constructs to map the sites of phosphorylation, we observed that a domain of 93 amino acids downstream of the ankyrin repeats incorporated the majority of P-32 in vivo. This sequence was also required for activation of the HES-1 promoter. In addition, we observed that hyperphosphorylated forms of the intracellular domain were more likely to interact with the transcriptional coactivator RBP. However, dephosphorylation experiments showed that the interaction between RBP and the intracellular domain of Notch was not dependent upon Notch1IC phosphorylation. These studies reveal that phosphorylation of the intracellular domain of the Notch receptor is a dynamic process during the events of Notch signal transduction.