Betacellulin is a member of the epidermal growth factor (EGF) family. These soluble proteins are ligands for one or more of the four receptor tyrosine kinases encoded by the erbB gene family (erbB-1/epidermal growth factor receptor (EGFR), neu/erbB-2/HER2, erbB-3/HER3 and erbB-4/HER4). While evidence suggests that betacellulin is a ligand for the EGFR, the ability of betacellulin to regulate other erbB family receptors has not been analysed. Previously we engineered derivatives of the mouse Ba/F3 hematopoietic cell line to ectopically express erbB family receptors, singly and in pairwise combinations. We have stimulated this panel of cell lines with betacellulin and two other EGF family members, EGF itself and neuregulin-beta (NRG-beta). In the cell lines expressing a single erbB family receptor, betacellulin not only stimulated EGFR tyrosine phosphorylation, but it activated erbB-4 as well. Furthermore, in the double recombinant Ba/F3 derivatives, betacellulin stimulated a complex pattern of receptor phosphorylation distinct from the patterns activated by NRG-beta and EGF. Moreover, betacellulin stimulated a complex pattern of interleukin-3 independence in the Ba/F3 derivatives distinct from those activated by NRG-beta and EGF. These data identify a novel receptor for betacellulin and establish that different EGF family ligands activate distinct patterns of receptor phosphorylation and coupling to cellular signaling pathways.