The renal collecting duct is a major target for the mineralocorticoid hormone aldosterone which acts to enhance electrogenic Na+ absorption. The cortical portion of the collecting duct displays a vigorous response to mineralocorticoids administered in vivo. The terminal, or inner medullary portion, does not usually display such a vigorous response; the reason for this difference is unknown. To explore one possible mechanism for this lack of response, we varied the conditions of culturing these cells and determined that serum inhibited the ability of aldosterone to enhance Na+ transport. By screening 11 peptides, we found that transforming growth factor (TGF)-beta 1 produced a concentration-dependent inhibition of the action of aldosterone. The action of TGF-beta 1 required at least several hours of incubation. Resistance to the action of aldosterone could be produced by preincubating the monolayers with TGF-beta 1 for a few hours; subsequent exposure to aldosterone for up to 48 h failed to stimulate Na+ transport. TGF-beta 1 did not produce a change in cell morphology or the content of DNA, ATP, or ADP; there was a small reduction in protein content. Pretreatment with cycloheximide failed to reproduce the TGF-beta 1 effect. The induction of resistance to mineralocorticoid hormone may play an important role in modulating the effects of aldosterone on Na+ homeostasis.