The present study was designed to examine the effects of a >30 kDa fraction of medium conditioned for 2 days by adult rat seminiferous tubules on inhibin secretion by cultured tubules, and on spermatogenesis and fertility of male rats. Inhibin secretion was assayed by adding the >30 kDa fraction to 5 cm segments of adult rat seminiferous tubules and measuring inhibin by radioimmunoassay at 2 day intervals. Fertility was assayed by injecting male rats daily for up to 45 days with the >30 kDa fraction and then mating them with a proestrus female, or by injecting for 15 days and mating them with two female rats. The assay used to evaluate the in-vivo effect of the >30 kDa fraction on the testis involved an assessment of frequencies of seminiferous tubule stages scored by transillumination on intact tubules. The addition of the >30 kDa fraction to the adult rat seminiferous tubules cultured for 2 days resulted in an inhibition of inhibin secretion into the medium. This effect was reversed when the fraction was removed and changed with fresh medium and cultured for a further 4 days. The >30 kDa fraction administered i.p. to adult male rats resulted in a low fertilization rate compared to control rats (67%) (P < 0.05). The assessment of frequencies of seminiferous tubule stages scored by transillumination showed an increased frequency of stage VI and decreased frequency of stages VII and VIII after treatment. The results of the present study provide additional evidence that local regulation of Sertoli cell function is mediated by a >30 kDa component or components secreted by adult seminiferous tubules which could arrest spermatogenesis. © 1995 Oxford University Press.