The effect of epidermal growth factor (EGF) on the production of immunoreactive inhibin by adult rat isolated seminiferous tubules in vitro has been investigated. EGF (0.1-1000 ng/ml) added to cultures of seminiferous tubules from adult rats caused a dose-dependent increase in inhibin content in the tubules without changing the amount secreted into the media. However, after continuous stimulation with EGF for periods in excess of 5 days, an increase in inhibin secretion was observed. In the presence of 10 and 100 ng FSH/ml, EGF (10 ng/ml) produced a further increment in the inhibin content of the tubules, but this effect was not found with FSH concentrations of 500 or 1000 ng/ml. EGF also increased the tubule content of inhibin after the addition of 100 μg dibutyryl cyclic AMP/ml but no effect of EGF was observed on the FSH- or dibutyryl cyclic AMP-induced secretion of inhibin into the medium. The effect of EGF on inhibin content in the tubules was partially suppressed by the addition of 4β-phorbol-12β-myristate-13α-acetate (20 ng/ml). Insulin (1-100 ng/ml) decreased basal inhibin secretion without changing the inhibin content of tubules and this effect was antagonized by EGF (10 ng/ml) with insulin doses of 1-50 ng/ml whereas, at 100 ng/ml, the effect of EGF on tubule inhibin content was reversed. The addition of EDTA (2 mmol/l) resulted in an inhibition of basal and EGF-induced inhibin production. These data demonstrate a stimulatory effect of EGF on inhibin production by isolated seminiferous tubules which is inhibited by insulin and phorbol esters, both stimulators of protein kinase C activity.