We have found that neuregulin-1beta (NRG-1beta) is expressed in the cardiac microvascular endothelium, and promotes the growth and survival of cardiac myocytes in culture through the activation of erbB2 and erbB4 receptor tyrosine kinases. In this study, we examined the role of NRG-1/erbB signaling in protection of cardiac myocytes from anthracycline-induced apoptosis in vitro to determine the coupling between erbB receptor subtypes and cytoprotective signaling. Treatment of neonatal rat ventricular myocytes with NRG-1beta inhibited daunorubicin-induced apoptosis as shown by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining for DNA fragmentation as well as flow cytometric quantification of apoptotic myocytes. Daunorubicin-induced activation of caspase-3 in cardiomyocytes was similarly inhibited by NRG-1beta. The phosphoinositol-3-kinase (PI3-kinase) inhibitor wortmannin prevented the effects of NRG-1beta on daunorubicin-induced apoptosis and activation of caspase-3. NRG-1beta treatment induced rapid activation of Akt/PKB that was inhibited by wortmannin, and adenoviral-mediated overexpression of a dominant-negative Akt prevented the protective effect of NRG-1beta. Akt activation by NRG-1beta was prevented by the tyrphostin AG1478, which we show inhibits erbB4 activation by NRG-1beta. In contrast, the erbB2-specific tyrphostin AG879 had no effect on NRG-1beta activation of Akt. Myocyte treatment with an activating antibody to erbB2 caused phosphorylation of erbB2, and led to activation of Erk but not Akt. Treatment with the erbB2 antibody had no effect on anthracycline-induced apoptosis. Thus, NRG-1beta protects against anthracycline-induced apoptosis via erbB4-dependent activation of the PI3-kinase/Akt pathway.