During a 24 h incubation of GH4C1 cells with phorbol esters or TRH, there was a decrease in the number of phorbol ester receptors (down-modulation). Whether this decrease in receptor number attenuated cellular responsiveness to subsequent challenge with phorbol esters was investigated. Cellular sensitivity to a phorbol ester-mediated biological response, the decrease in binding of epidermal growth factor, was compared in control and down-modulated cells. This phorbol ester-mediated event is closely associated with phorbol ester receptor occupancy. During a 48 h exposure to phorbol esters, GH4C1 cells became refractory to the effect of epidermal growth factor binding. This time course was similar to that for the loss of phorbol ester receptors. However, when cells were down modulated by pretreatment with phorbol ester or thyrotropin-releasing hormone and then (re)challenged with phorbol ester, no differences in dose-response characteristics were observed between control and down-modulated cells. Phorbol ester receptor down-modulation apparently does not affect cellular responsiveness to phorbol esters, at least when decreased epidermal growth factor binding is used as the marker for the phorbol ester-mediated event.