When added to cultured opossum kidney cells, IGF-I is internalized and transported to distinct intracellular compartments that depend on the cell location within the monolayer. In resting cells away from the periphery of the monolayer, IGF-I is internalized by a clathrin coated pit pathway and delivered to the endosomal compartment. In contrast, cells growing at the edges of a monolayer or an experimental wound internalize IGF-I by an alternative route which rapidly delivers IGF-I to the nucleus. Similarly to IGF-I, IGFBP-3 is also internalized and accumulates in the endosomal compartment in resting cells whereas it is targeted to the nucleus in proliferating cells. IGFBP-3, which contains a putative nuclear targeting signal, may act as a carrier for IGF-I nuclear transport. The transport of IGF-I and IGFBP-3 to two different compartments may influence their biological activity.