Neu differentiation factor (NDF or heregulin) elevates tyrosine phosphorylation of the ErbB-2 receptor tyrosine kinase, and it was, therefore, thought to function as a ligand of this receptor. However, several lines of evidence raised the possibility that the interaction between NDF and ErbB-2 involves another molecule, which belongs to the family of epidermal growth factor receptors. To address this question we constructed soluble chimeric proteins between alkaline phosphatase and the extracellular domains of ErbB-2 and either ErbB-3 or ErbB-4, two newly recognized members of the epidermal growth factor receptor family. Using the soluble proteins we found that beta isoforms of NDF specifically bind to the ErbB-3 and ErbB-4 receptors but not to the soluble ErbB-2 protein. When ectopically expressed in monkey fibroblasts, the full-length ErbB-3 and ErbB-4 receptors conferred specific binding to NDF. In these cells ErbB-3 displayed lower ligand binding affinity than ErbB-4, but like the latter receptor it preferred to bind the beta isoform over the alpha class of NDFs. These results indicate that both ErbB-3 and ErbB-4 function as physiological receptors of all NDF isoforms and suggest that a still unknown ligand of ErbB-2 exists.