The factors that regulate growth and function of the human adrenal gland during intrauterine development and thereafter are ill defined. Whereas others have reported that adrenocorticotropic hormone (ACTH) augments the inhibitory effect of transforming growth factor-β (TGF-β) on growth of fetal zone (FZ) cells of the human fetal adrenal, we recently found that ACTH interferes with TGF-β's inhibition of growth of fetal adrenal neocortex cells. In this study we sought to assess independently the effects of TGF-β in the absence and presence of ACTH on growth of FZ cells. TGF-β, in a time- and dose-dependent manner, inhibited growth (i.e., [3H]thymidine incorporation) of FZ cells. ACTH (Cortrosyn), at 90 pM to 90 nM was found to interfere with the TGF-β inhibition of FZ growth. ACTH 1-24 and human ACTH 1-39, both from Sigma Chemical, also were found to blunt the response of FZ cells to TGF-β. Growth inhibition due to TGF-β action and the reversal by ACTH of TGF-β effects on FZ cell growth were confirmed by the results of immunohistochemical analyses of 5'-bromo-2'-deoxyrudine incorporation into nuclei of FZ cells and by indirect evaluations of cell numbers. Both forskolin (10 μM and dibutyryl) adenosine 3',5'-cyclic monophosphate (1 nM), but not phorbol 12-myristate 13-acetate (1 or 100 mM), were able to mimic ACTH actions in blunting the inhibitory effects of TGF-β on DNA synthesis. We conclude that ACTH, possibly via activation of adenylate cyclase, interferes with, rather than augments, the growth-inhibitory effect of TGF- β on FZ cell growth.