Using two-site enzyme immunoassays, we measured protein levels of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and heparin-binding epidermal growth factor (HB-EGF) in adult rat brain, and compared them with the phosphorylation levels of their receptor (ErbB 1). There were significant variations in the brain distributions of each ErbB I ligand. Among these ErbB1 ligands, HB-EGF protein levels were higher than those of TGF alpha and those of EGF were the lowest. TGF alpha protein was relatively enriched in the midbrain regions, while HB-EGF levels were most abundant in the cerebellum. Protein distributions of the EGF family members were discordant with previously reported mRNA distributions. In addition, there was significant basal ErbB I phosphorylation detected with the largest amount of activation in the midbrain. These observations suggest that the activation of brain ErbB1 involves post-translational regulation of multiple EGF family members in a region-specific manner. (C) 2005 Elsevier Ireland Ltd. All rights reserved.