The adult liver is the main source of circulating insulinlike growth factors (IGFs) and their serum binding proteins (IGFBPs) including the acid-labile subunit (ALS), a component of the ternary binding protein complex. Within the liver, the biosynthesis of individual proteins has been attributed to different cell populations, e.g., that of ALS to hepatocytes and that of IGFBP-3 to nonparenchymal cells. Ligand and immunoblotting as well as Northern blotting analyses were used to study synthesis of IGFBPs and their hormonal regulation in cultured adult rat hepatocytes, Kupffer cells (KCs), and cocultures. In hepatocytes, synthesis of IGFBP-1, -2, and -4 was observed; insulin and IGF-I decreased that of IGFBP-1, and -2 while increasing that of IGFBP-4. KCs synthesized IGFBP-2, and -3, insulin and IGF-I showing no effect. In cocultures, however, synthesis of IGFBP-3 was stimulated by insulin and IGF-I. By immunocytochemistry IGFBP-3 biosynthesis was localized to KCs exclusively. When pore membranes were used for separation of hepatocytes and KCs in coculture, this insulin-stimulatory action on IGFBP-3 synthesis was preserved. Growth hormone (GH) did not affect biosynthesis of IGFBPs. Expression of ALS was localized in hepatocytes only. Insulin, IGF-I, and GH increased ALS expression. It can be concluded that biosynthesis of individual IGFBPs and of ALS are compartmentalized in adult rat liver and are distinctly regulated by insulin, IGF-I, and GH. The insulin-dependent stimulation of IGFBP-3 synthesis in KCs appears to require a diffusable mediator derived from hepatocytes.