In most cell types two classes of epidermal growth factor (EGF) receptors can be found: a major class that binds EGF with relatively low affinity and a minor class that binds with very high affinity. Structure–function studies have shown that mutations at amino acid 47 in the EGF molecule severely reduce its affinity for the EGF receptor but do not cause preferential binding to one or the other subclass of receptors. Using three EGF derivatives with a mutation at amino acid 47 (Ser-47, Leu-37-Tyr-47, and Val-47), we have investigated the relative contribution of the two receptor subclasses to the EGF-dependent mitogenic response. We show that mitogenicity correlates exclusively with occupancy of the high-affinity receptor and that full occupancy of this subclass is required for maximal stimulation. In addition we demonstrate that for the EGF-Val-47 analogue this requirement can be abrogated and half-maximal biological activity reached with a high-affinity receptor occupancy of only 8%. While the rate of internalization did not significantly differ between EGF-Val-47 and native mEGF, the analogue was much more resistant to degradation by cellular proteases and, after binding and receptor-mediated internalization, was released into the medium predominantly in an intact form. We propose that the increased mitogenicity of EGF-Val-47 is due to its prolonged half-life, resulting in continued occupancy of the high-affinity EGF receptor.