Treatment of pituitary GH4C1 cells with epidermal growth factor (EGF) caused up to a 60% reduction in the amount of [3H]MeTRH bound to specific TRH receptors. The effects of EGF were first detectable after a 2-h incubation and maximal by 24-72 h. EGF elicited a half-maximal response at 0.03 nM. Equilibrium binding analysis was performed on intact cells that had been incubated with or without 10 nM EGF for 96 h. EGF decreased the apparent number of TRH receptors (maximum binding = 0.36 vs. 0.58 pmol/mg protein for EGF-treated and control cells, respectively) without altering the apparent affinity (dissociation constant = 6.4 vs. 7.4 nM). The effects of EGF on TRH receptors were reversible. When EGF was removed from the medium, TRH receptors returned to control levels within 48 h. To assess whether the reduction of TRH receptors was functionally important, the ability of TRH to stimulate phospholipid turnover was measured in cells with a normal complement of TRH receptors and in cells that had been treated with EGF for 72 h to reduce TRH receptor density. EGF significantly blunted the ability of TRH to stimulate release of inositol phosphates from metabolically labeled cells. TRH increased inositol monophosphate accumulation 6.3-fold in control cultures and 2.0-fold in EGF-treated cells. These data show that EGF regulates the concentration of TRH receptors on pituitary GH4C1 cells and the responsiveness of the cells to TRH.