The saturated fatty acid palmitate alters normal cell function via disruption of cell signaling, and this effect has been implicated in the end-organ damage associated with dyslipidemia. Neuregulin-1beta (NRG-1beta) is a growth and survival factor in cardiac myocytes. We tested the hypothesis that palmitate alters NRG-1beta signaling and biology in isolated neonatal rat cardiac myocytes. Palmitate treatment inhibited NRG-1beta activation of the PI3-kinase/Akt pathway in myocytes. We found that the pro-apoptotic activity of palmitate was increased by NRG-1beta treatment. The effects of palmitate on NRG-1beta signaling and survival were reversed by the mono-unsaturated fatty acid oleate. Under control conditions NRG-1beta decreases p53 expression in myocytes. In the presence of palmitate, NRG-1beta caused an increase in p53 expression, bax multimer formation, concurrent with degradation of mdm2, a negative regulator of p53. Thus in the presence of palmitate NRG-1beta activates pro-apoptotic, rather than pro-survival signaling in cardiac myocytes.