The exact nature of how the insulin molecule interacts with the insulin receptor is obscure although chimeric receptors have shown that the ligand specificity of the insulin receptor and the IGF-I receptor (i.e. the sequences that discriminate between insulin and insulin-like growth factor I) reside in different regions of a common binding site and that the N-terminal 68 amino acids of the insulin receptor are involved in conferring specificity for insulin on this receptor (Kjeldsen, T., Andersen, A. S., Wiberg, F. C., Rasmussen, J. S., Schäffer, L., Balschmidt, P., Møller, K. B., and Møller, N. P. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4404-4408). Using chimeric insulin/IGF-I receptors to elucidate how the insulin receptor interacts with the insulin molecule we identified phenylalanine 39 of the insulin receptor as a major contributor in determining the receptor specificity for insulin, increasing insulin affinity 15-fold when replacing the corresponding amino acid in the insulin-like growth factor I receptor. Furthermore, replacement of the insulin receptor amino acid phenylalanine 39 with the corresponding IGF-I receptor amino acid, serine 35, decreased insulin affinity 8-fold.