The role of transforming growth factor beta (TGF-beta) on the functions of pig Leydig cells cultured in a chemically defined medium was investigated. TGF-beta reduced the number of hCG receptors, without modification of the binding affinity, and reduced the cAMP and testosterone response to this hormone. These effects were dose-dependent. The minimal effective dose was 10 pg/ml (4 X 10(-13) M) and half-maximal inhibition for the three effects was observed at about 100 pg/ml. At maximal effective concentration (1 ng/ml), the inhibitory effect was time-dependent, the first effects were observed after a lag period of 12 h and the maximal effect after 72 h. At maximal concentrations TGF-beta reduced by 70% the number of hCG receptors and the steroidogenic response to this hormone and reduced by 50% the cAMP response to hCG. Moreover, TGF-beta also reduced the cAMP response to forskolin and the steroidogenic effects of this diterpene and 8-Bromo-cAMP. In contrast, the conversion of exogenous pregnenolone to testosterone was increased in TGF-beta-treated cells. The inhibition of Leydig cell steroidogenesis can be dissociated from any effect on cell proliferation and is related to modifications located at the membrane level and beyond cAMP formation, but before pregnenolone formation. The results suggest that in the testis, as in other steroidogenic tissues, TGF-beta may play a role in the development and maintenance of differentiated function.