In order to define insulin-like growth factor 1(IGF-1) binding sites on IGF-receptor 1 (IGF-R1), the interactions of IGF-1 and IGF-R1 was verified in the yeast two-hybrid system. Seven mutants IGF-R1 were generated by site-directed in vitro mutagenesis, then the interactions of IGF-1 and various mutants IGF-R1 were quantified to identify the gain-of-function mutation in the yeast two-hybrid system by assaying the β-galactosidase activity. The results confirmed the binding data of IGF-1 to IGF-R1 and suggested that the residues N237 and T238 on IGF-R1 play a crucial role in binding to IGF-1.