ErbB2, ErbB3 and ErbB4 are members of the Epidermal Growth Factor Receptor (EGFR) sub-family of Receptor Tyrosine Kinases (RTKs). Neuregulin-1 (NRG-1) is a ligand of ErbB3 and ErbB4 receptors. NRG-1-induced ErbB2/ErbB3 or ErbB2/ErbB4 heterodimerization, followed by receptor phosphorylation, plays multiple biological roles. To precisely determine the phosphorylation status of each ErbB receptor in ErbB2/ErbB3 and ErbB2/ErbB4 heterodimers, an immunoprecipitation-recapture of the ErbB receptors was performed to exclude any co-immunoprecipitated heterodimer partners from cells with co-expression of ErbB2/ErbB3, ErbB2/ErbB4, or ErbB2/ErbB4D843N, a kinase-inactive ErbB4 mutant, in which the aspartic acid at 843 (D843) was replaced by an asparagine (N). Here, we provide direct biochemical evidence that ErbB2 was only trans-phosphorylated by ErbB4, but not by ErbB3 or ErbB4D843N. By contrast, ErbB3, ErbB4 and ErbB4D843N were trans-phosphorylated by ErbB2 in the co-transfected cells. Therefore, we conclude that trans-phosphorylation, but not cis-phosphorylation occurred between ErbB2/ErbB3 and ErbB2/ErbB4 heterodimer partners by NRG-1 stimulation.