Two lines of EL4 mouse thymoma cells, one which responds to phorbol ester tumor promoters with production of T-cell growth factor and inhibition of proliferation and one which does not respond, have been examined for the presence of specific phorbol ester-binding components. Both lines displayed saturable specific binding as determined by using [20-3H]phorbol 12, 13-dibutyrate in a whole-cell-binding assay. Specific binding in each line was maximal within 10 min at 37° but rapidly decreased to about 30% within 30 min. Maximal binding at 4° was reached after 60 min and was stable for at least 8 hr; however, this level was only about 30% of the maximum obtained at 37°. Saturation of the specific binding after 3 hr at 4° occurred at a concentration (30 to 50 nm) which is consistent with that yielding maximal T-cell growth factor production in the responding line but greater than that at which inhibition of proliferation was detected in those cells. Scatchard analysis of these data was consistent with the existence of a single class of binding sites with a Kd of 11 ± 0.6 (S.D.) nm for the T-cell growth factor-producing cells and 18 ± 1.9 nm for the nonproducing line. The numbers of sites per cell measured in the responding and nonresponding lines were 5.6 ± 1.3 × 104 and 7.1 ± 0.2 × 104, respectively. Competition by a series of phorbol esters for [20-3H]phorbol 12,13-dibutyrate binding in the responding line showed the same order of potency as production of T-cell growth factor and inhibition of proliferation by the analogs. These data support identification of the phorbol ester-binding component in the responding cells as the receptor mediating T-cell growth factor production. The presence of binding components in unresponsive cells in numbers and with characteristics indistinguishable from those of responsive cells suggests that the failure to respond is due to modification of a step which succeeds the initial binding event.