The insulin/IGF system plays a critical role in embryo growth and development. We have investigated the expression of insulin receptor (IR) and IGF-I receptor (IGF-IR) and the activation of their downstream pathways in rabbit 6-d-old blastocysts. IR was expressed in embryoblast (Em, inner cell mass) and trophoblast (Tr) cells, whereas IGF-IR was localized mainly in Em. Isoform A (IR-A) represents the main insulin isoform in blastocysts and was found in Em and Tr cells. IR-B was detectable only in Tr. IR/IGF-IR signaling pathways were analyzed after stimulation with insulin (17 nm) or IGF-I (1.3 nm) in cultured blastocysts. Insulin stimulated Erk1/2 in Em and Tr and Akt in Tr but not in Em. IGF-I activated both kinases exclusively in Em. The target genes c-fos (for MAPK kinase-1/Erk signaling) and phosphoenolpyruvate carboxykinase (PEPCK, for PI3K/Akt signaling) were also specifically regulated. Insulin down-regulated PEPCK RNA amounts in Tr by activation of the phosphatidylinositol 3-kinase/Akt pathway. Expression of c-fos by insulin and IGF-I was different with respect to time and fortitude of expression, mirroring again the specific IR and IGF-IR expression patterns in Em and Tr. Taken together, we show that IGF-I acts primarily mitogenic, an effect that is cell lineage-specifically restricted to the Em. By contrast, insulin is the growth factor of the Tr stimulating mitogenesis and down-regulating metabolic responses. As soon as blastocyst differentiation in Em and Tr has been accomplished, insulin and IGF-I signaling is different in both cell lineages, implying a different developmental impact of both growth factors.