Phorbol ester binding to intact GH4C1 cells, a continuous strain of rat pituitary cells, was measured using [3H]phorbol 12,13-dibutyrate. The binding was saturable; Scatchard analysis indicated one class of high-affinity binding sites (Kd, 11 nm) with 6.4 pmol [3H]phorbol 12,13-dibutyrate bound per mg cell protein at saturation. The relative binding affinities for other phorbol esters and analogs were similar to those demonstrated for binding to homogenates of chick embryo fibroblasts and mouse skin, as well as for tumor promotion in vivo. A close correlation was shown to exist between the binding affinities of these derivatives and their potencies for inducing biological responses in GH4C1 cells, such as decreases in binding of epidermal growth factor and thyrotropin-releasing hormone. A distinctive new finding is the down modulation of phorbol ester binding sites on GH4C1 cells by both homologous and heterologous ligands. Prolonged exposure to phorbol esters or thyrotropin-releasing hormone produced a loss of available [3H]-phorbol 12,13-dibutyrate binding sites with a maximal decrease to about 20% of control after 24 hr of treatment. Scatchard analysis indicated that the decrease in binding was due to a loss of receptors with no change in affinity. The biological significance of phorbol ester receptor down modulation is not yet known; however, it may represent a mechanism for attenuating cellular responsiveness to phorbol esters in their continued presence.