The c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A or 2B mutation can transform NIH3T3 cells with high efficiencies as a consequence of its constitutive activation. The MEN2A mutation induces ligand-independent homodimerization of the Ret protein on the cell surface while the MEN2B mutation appears to alter the catalytic activity without dimerization. In the present study, we investigated the role of tyrosine residues present in the kinase domain for the transforming activity of the mutant Ret proteins. Substitution of phenylalanine for tyrosine 905 (Y905F) that corresponds to tyrosine 416 of the Src protein abolished the transforming activity of Ret with the MEN2A mutation (MEN2A-Ret) but not with the MEN2B mutation (MEN2B-Ret). On the other hand, the transforming activity of MEN2B-Ret but not MEN2A-Ret significantly decreased by changing tyrosine 864 or 952 to phenylalanine. In addition, double mutations of these tyrosines (Y864/952F) completely abolished the activity of MEN2B-Ret. The Y905F and Y864/952F mutations resulted in severe impairment of the kinase activity of MEN2A-Ret and MEN2B-Ret, respectively. These results thus indicated that tyrosine residues essential for the transforming activity are different between MEN2A-Ret and MEN2B-Ret.