Alterations that affect the ectodomain of receptor tyrosine kinases are often associated with constitutive activation of the enzymic activity of the mutant cell-associated receptor. Since the ectodomain of the ErbB2 receptor tyrosine kinase has been detected as a soluble fragment in the culture supernatant of cells and serum from patients with advanced breast cancer, the possible presence of cell-associated truncated forms of ErbB2 in cancer cells was investigated. Several cell-bound N-terminal truncated forms of ErbB2 were identified in breast cancer cells overexpressing this receptor. The presence of the truncated fragments was independent of lysosomal/proteasomal activity, indicating that classical receptor tyrosine kinase degradation systems were not involved in the N-terminal cleavages. The presence of these truncated forms of ErbB2 was up-regulated by protein kinase C and neuregulin; and down-regulated by phosphatidylinositol 3-kinase, and monoclonal antibodies that target the ectodomain of ErbB2, indicating that N-terminal cleavages of ErbB2 were regulated by multiple mechanisms. The truncated fragments were tyrosine-phosphorylated under resting conditions, and associated with the signalling intermediates Shc and Grb2. It is therefore likely that these truncated forms may be endowed with constitutive activity that allows them to permanently signal.