Sertoli cell aromatase activity is high in very young animals and declines throughout pubertal development. Little is known about the regulatory factors which might be involved in the pronounced decline suffered by this enzymatic activity. In this paper we show that estradiol production in Sertoli cells is dependent on cell density in the culture and that chronic stimulation with hormones can decrease estradiol acute response to FSH. In 8-day-old Sertoli cells cultured at low density (LD: 7.1 +/- 0.3 micrograms DNA), estradiol production was 151 +/- 11 pgE2/micrograms DNA, while in those cultured at high density (HD: 30.3 +/- 0.6 micrograms DNA), production was 30 +/- 5 pgE2/micrograms DNA. Similar results were obtained in 20-day-old Sertoli cell cultures (LD: 57 +/- 4 pgE2/micrograms DNA vs HD: 26.0 +/- 0.6 pgE2/micrograms DNA). On the other hand, treatment of Sertoli cell cultures (8- and 20-day-old) for 96 h, with FSH (100 ng/ml), EGF (50 ng/ml), insulin (10 micrograms/ml) and IGF-I (50 ng/ml) at different densities resulted mostly in inhibition of aromatase activity. The effect caused by FSH was apparently not related to desensitization as aromatization with dbcAMP could not overcome the decreased ability of these cells to produce estradiol. The effect caused by EGF was observed in 8-day-old Sertoli cells cultured under high density conditions. Marked inhibition was observed with insulin and IGF-I in 8-day-old Sertoli cell cultures. Considering previous reports indicating a decrease in Sertoli cell aromatase activity with age, our results suggest a potential role for FSH, EGF, insulin and IGF-I on the Sertoli cell differentiation process which occurs throughout pubertal development.