The enhancement of DNA synthesis by epidermal growth factor (EGF) in cultured human fibroblasts is demonstrable 24 hr after incubation of the cells at 37 degrees C with very low concentrations (0.83 nM) of the hormone for very short periods (30 min) followed by thorough washing of the cells to remove the free hormone in the medium. This effect must result from persistent, extraordinarily tight binding of the hormone to surface receptors, because the addition of specific, purified anti-EGF IgG as late as 8 hr after initial hormone exposure can completely reverse the biological effects of the hormone. This causes only a slight (but significant) increase in the rate of dissociation at 37 degrees C of the cell-bound (125)I-labeled EGF at low occupancy. Together with the fact that in the presence or absence of antibody virtually all of the demonstrable cell-bound (125)I-labeled EGF can be shown to dissociate from the cell during a period as short as 2-3 hr, the data suggest the possibility that the biological effects of this hormone may be mediated by occupation of only a negligible fraction of very high affinity binding sites. Thus, the processes of hormone internalization, degradation, and "down regulation" may be irrelevant to the effects of the hormone on DNA synthesis. For this effect the crucial and limiting processes appear to be strictly related to the continuous and persistent occupation of cell surface receptors.