T o identify receptor-associated proteins that may contribute to the specificity of insulin and IGF-I signaling responses, a mouse embryo library was screened using the yeast two-hybrid system. Multiple receptor-interactive clones encoding the SH2 domain of the adapter protein Grb10 were isolated. Subsequent cloning of the full-length Grb10 sequence from a mouse fat cDNA library defined a previously unknown Grb10 variant, that appears to be the predominant isoform in mouse tissues. Receptor-deficient R- cells (fibroblasts from mice with homologous disruption of the IGF-I receptor gene) and transfected R- cells expressing either insulin receptors (R-IR cells) or IGF-I receptors (R+ cells) were used to investigate the specificity of Grb10 interaction with the two related receptors. Hormone-activated insulin receptors in R-IR cells coprecipitated with three species, all recognized as Grb10 isoforms by specific Grb10 antibody. Under the same conditions, Grb10 was essentially undetectable in IGF-I receptor immunoprecipitates from stimulated R+ cells. Grb10 association with insulin receptors was maximal at 10 nM insulin stimulation and sustained from 5-10 min after hormone stimulation in R-IR cells. In conclusion, Grb10 interacts preferentially with insulin vs. IGF-I receptors in intact cells and, thus, may have a role in mediating insulin receptor-specific cellular responses.