The four members of the EGF receptor family are capable of homomeric as well as heteromeric interactions. HER-2/neu (erbB-2) dominates as the preferred coreceptor that ampli®es mitogenic signaling. An alternative HER-2/neu product, herstatin, consists of a segment of the ectodomain of p185HER-2 and an intronencoded C-terminus. Recombinant herstatin was found to bind with nM a nity and inhibit p185HER-2. To further examine the impact on receptor activity, herstatin was expressed with various receptor tyrosine kinases. In CHO cells that overexpressed HER-2, herstatin caused a sevenfold inhibition of colony formation that corresponded to a reduction in the tyrosine phosphorylation of p185HER-2. Herstatin also prevented HER-2 mediated transactivation of the kinase impaired HER-3 as re¯ected in transphosphorylation of HER-3 and heteromers between HER-2 and HER-3. In EGF receptor-overexpressing cells, EGF induction of receptor dimerization and tyrosine phosphorylation were reduced more than 90%, and receptor down-regulation as well as colony formation were also suppressed by coexpression with herstatin. Inhibition was selective for the EGF receptor family since herstatin expression did not reduce tyrosine phosphorylation mediated by the FGF receptor-2 or by insulin-like growth factor -1. Herstatin bound to the EGF receptor as well as to p185HER-2 in pull-down assays suggesting that complex formation may be involved in receptor inhibition. Our ®ndings indicate that herstatin has the capability to negatively regulate combinations of interactions between group I receptor tyrosine kinases that confer synergistic growth signals. Oncogene (2001) 20, 5199 ± 5209.