Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane precursor (proHB-EGF). Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C, respectively). We have previously shown that HB-EGF-C is translocated from the plasma membrane into the nucleus, where it interacts with the transcription repressor, PLZF. Here we characterize the amino acid residues of the cytoplasmic domain of proHB-EGF on cell surface distribution and the interaction of HB-EGF-C with PLZF. The cytoplasmic domain contains three characteristic clusters with charged amino acids. Generation of various mutants of proHB-EGF showed that the arrangement of the charged amino acids in the cytoplasmic domain regulates the distribution of proHB-EGF at the plasma membrane but does not regulate proHB-EGF processing and internalization of HB-EGF-C. Further, the charged amino acids are also required for HB-EGF-C-PLZF interaction. These results indicate that the cytoplasmic domain of proHB-EGF is a multifunctional domain.