Human insulin-like growth factor-1 (hIGF-1) contains seven positively charged residues in the A-, C-, and D-regions that are not present in similar positions in insulin. To determine whether these residues contribute to receptor binding specificity for the insulin-like growth factor-1 receptor (IGF-1R) relative to the insulin receptor (IR) we examined the binding of hIGF-1 analogs in which these residues have been replaced with either alanine or the corresponding residue of insulin. To improve expression and facilitate purification we employed insulin-like growth factor (IGF-1) analogs modified with an N-terminal 8-amino acid epitope. This additional epitope did not alter receptor binding specificity. Alanine substitution for the positively charged residues in the C- and D-regions of IGF-1 led to 15- and 10-fold losses, respectively, in binding potency for the human IGF-1R, but they increased the potency of binding to the human IR 29- and 6-fold, respectively. In contrast, substitution of the positively charged side chains in the A-region with the corresponding uncharged residues of insulin had little effect on binding to either receptor. These data suggest that the positive charges in the C- and D-regions of IGF-1 contribute significantly to the binding preference of the IGF-1R for IGF-1. In complementary experiments using chimeric receptors we have also shown that the IGF-1 receptor elements required to discriminate in favor of the positive charges in the C- and D-regions are contained in the N-terminal 283 amino acids of the alpha-subunit. Insulin receptor elements that discriminate against these charges are within the N-terminal 225 amino acids of its alpha-subunit.