Rates of production of androgen binding protein (ABP) declined during culture of purified Sertoli cell-enriched aggregates in serum-free minimal essential medium (MEM). This decline was partially prevented by the addition of testosterone to the medium, but not by 17 beta-estradiol addition. The androgen effect was greater in Sertoli cell aggregates in co-culture with peritubular cells than in Sertoli cell preparations depleted of peritubular cells. Initial rates of ABP production by purified Sertoli cell-enriched aggregates were also sustained when preparations were cultured in MEM supplemented with conditioned medium in which peritubular cells had been previously maintained. We interpret data presented to indicate that the stimulatory effects of peritubular cells on functions of Sertoli cells in co-culture, specifically sustained rates of ABP production, are mediated at least partially by component(s) synthesized by peritubular cells and secreted into the medium. We offer the hypothesis that androgens may influence Sertoli cells at two levels: (1) a direct action on Sertoli cells; (2) and indirect influence on Sertoli cells mediated by a direct action on peritubular cells, resulting in the formation of product(s) which modulate the rates of ABP synthesis.