During a 24-hr incubation of GH4C1 cells with phorbol esters or thyrotropin-releasing hormone, there is a decrease in the number of phorbol ester receptors (down-modulation). The purpose of this study was to investigate whether this decrease in receptor number attenuated cellular responsiveness to subsequent challenge with phorbol esters. Accordingly, cellular sensitivity to a phorbol ester-mediated biological response, namely 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, and it is therefore an effect for which altered dose-response characteristics correlating with alterations in the number of phorbol ester receptors could be anticipated. In fact, during a 48-hr exposure to phorbol esters, GH4C1 cells become refractory to the effect on epidermal growth factor binding. This time course is similar to that for the loss of phorbol ester receptors. However, when cells are down modulated by pretreatment with phorbol ester or thyrotropinreleasing hormone and then (re)challenged with phorbol ester, no differences in dose-response characteristics were observed between control and down-modulated cells. We therefore conclude that phorbol ester receptor down-modulation 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.