Insulin-like growth factor-I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of ACTH and angiotensin II (AII), as well as the secretion of IGF-I and its binding proteins (IGFBPs). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days of treatment with AII (1μm) or ACTH (10 nm) the number of stained cells increased by 5- and 14-fold respectively. In all cases the staining was specific, since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I into the medium, evaluated by a specific radioimmunoassay, was increased two- and sevenfold by AII and ACTH respectively. Using the method of Western ligand blotting, the major form of IGFBP secreted by control adrenal cells was found to be a 38–42kDa doublet protein. Two minor forms with apparent molecular weights of 28–31 kDa and 24kDa have also been identified. Following acid—ethanol extraction of the conditioned medium, all the IGFBPs were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent, AII pretreatment increased the 38–42kDa IGFBP by several fold, decreased the 28–31 kDa IGFBP and had no effect on the 24kDa IGFBP. In conclusion, these results demonstrate (i) that bovine adrenal cells contain IGF-I-like immunoreactive material, (ii) that the stimulatory effects of ACTH and AII on IGF-I secretion by bovine adrenal cells are due mainly to an increase in the number of IGF-I-producing cells and (iii) that ACTH and AII modulate the secretion of IGFBP by adrenal cells. Although the roles of IGFBPs have not been defined in adrenal cells, they are capable of modulating the biological action of IGFs in other cell cultures. Regulation of both IGF-I and its binding proteins by the two specific hormones ACTH and AII suggests important roles for these binding proteins in modulating the action of IGF-I in bovine adrenal cell function.