We reported previously that transforming growth factor β (TGF-β) does not influence the proliferation of bovine adrenocortical cells but is a very potent inhibitor of their steroidogenic functions (Feige et al.: Journal of Biological Chemistry 262: 13491-13495, 1987). In the present study, we addressed the question of whether these cells modify the synthesis of their extracellular matrix (in particular of fibronectin) in response to TGF-β 1, similarly to the changes observed in cell types whose growth is modified by this factor (eg, fibroblasts). Immunofluorescence studies using anti-fibronectin antibodies revealed that TGF-β 1, treatment in serum-free medium induced the formation of fibronectin-containing fibrils associated with adrenocortical cells. Metabolic labeling of adrenocortical cells with [35 S]-methionine showed that fibronectin synthesis and secretion were highly stimulated by low concentrations of TGF-β 1. Half-maximal stimulation was observed for TGF-β 1 concentrations in the range of 0.1 to 0.5 ng/ml and maximal stimulation reached 35-fold over control at the concentration of 2 ng/ml. The earlier detectable effect was observed after 8 h of treatment (6-fold stimulation) and the maximal increase was reached after 24 h of treatment. Stimulation of adrenocortical fibronectin synthesis by TGF-β 1 appeared to imply a transcriptional event since it was no longer observed in the presence of DRB, a potent inhibitor of RNA polymerases, and because the level of fibronectin mRNA was stimulated under TGF-β 1 treatment. Taken together, these results indicate that the increased expression of fibronectin is not closely related to growth-regulatory effects of TGF-β 1 since it is also observed in adrenocortical cells, whose proliferation is unaffected by TGF-β 1.