We have examined, by use of a hybrid insulin/insulin-like growth factor-I analog and chimeric insulin/type I insulin-like growth factor receptors, the interplay between ligand and receptor structure in determining the affinity and specificity of hormone-receptor interactions in the insulin and insulin-like growth factor-I systems. Our findings, obtained through the study of radiolabeled peptide binding to detergent-solubilized full-length receptors and to soluble truncated receptors, show that (a) the two-chain hybrid analog exhibits significant cross-reactivity with both receptor systems, (b) the exchange of appropriate domains in chimeric receptors enhances the receptor binding affinity of the analog by 3.5-21-fold, and (c) the affinity of the hybrid analog for the chimeric receptors actually exceeds that of either natural insulin or natural insulin-like growth factor-I. We conclude that the specificity-conferring domains of the insulin and type I insulin-like growth factor receptors reside in different regions of a common binding site, and that the exchange of domains between pairs of related hormones and between pairs of related receptors can yield new ligand-receptor systems with significantly altered affinities and selectivities.