The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivophosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase.