The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.