Several inhibitors of EGF receptor (EGFR) tyrosine kinase activity have been developed that compete with ATP at its binding site such as the quinazolines PD 153035 and ZD 1839 or the 4,5-dianilino-phthalimides DAPH1 and DAPH2. When tested on human A431 cells, the quinazolines completely blocked EGF-induced receptor phosphorylation at 100 nM, whereas it was inhibited by DAPH1 and DAPH2 by only 20% at 3 microM. Quinazoline-treated A431 as well as tumor cells expressing less EGFR (A549, MDA MB 231, and T47D) bound 3- to 6-fold more (125)I-labeled EGF than untreated intact control cells. Scatchard analysis revealed the disappearance of low- and high-affinity EGFR on A431 cells upon PD 153035 treatment. A single receptor class of intermediate ligand binding affinity emerged and its number corresponded to the sum of the two classes. DAPH1 and DAPH2 did not change ligand binding properties of EGFR. PD 153035 exerted the most potent effects on EGF binding to A431 or on inhibiting EGF-stimulated growth of rat MTLn3 cells at low ligand concentrations. Cross-linking of EGFR on PD 153035-treated A431 cells indicated the formation of inactive dimers that further increased upon addition of EGF. Chemical cross-linking of (125)I-labeled EGF to PD 153035-treated A431 cells revealed increased binding to monomeric and dimeric EGFR. Thus, the quinazolines sequestered EGFR plus the ligand into inactive receptor/ligand complexes. This novel mode of action of quinazoline tyrosine kinase inhibitors may be the basis for their extraordinary potency especially in conditions when the ligand is present in limiting amounts.