Specific biological responses to the erbB3-erbB4 ligand heregulin (HRG) have been postulated to be due to the formation of heterodimers of those receptors with erbB2. To test the role that erbB2 plays in the response to HRG in a human breast carcinoma cell line, antisense oriented erbB2 was stably transfected into AU565 cells. In the absence of HRG, inhibition of erbB2 expression slowed cell growth, leading to accumulation of cells in the G2/M phase, and suppressed colony growth in soft agar. Low concentrations of HRG induced cell proliferation in both the erbB2-nonexpressing cells and the parental AU565 cells. In contrast, high concentrations of HRG failed to induce differentiation of the erbB2-nonexpressing cells as compared with the parental cells. ErbB3 expression was significantly decreased in the erbB2 nonexpressing cells. ErbB3 was constitutively tyrosine phosphorylated in both the parental AU565 cells and in the erbB2 nonexpressing cells. HRG further increased tyrosine phosphorylation of erbB3 with a maximum response at 1 ng/ml of HRG in erbB2 nonexpressing cells, as compared with 10 ng/ml of HRG in AU565 cells. This finding suggested that the biochemical responsiveness of erbB3 to HRG was changed, but not abrogated, by inhibition of erbB2 expression. These results suggest that inhibition of erbB2 expression modulates, but does not abolish, HRG mediated signal transduction pathways in a human breast cancer cell line.