1. Inhibition of uncontrolled epidermal growth factor receptor (EGFR) is one of the approaches for the treatment of breast and lung cancers. We designed oligopeptides consisting of amino-acid sequences of the major (Y1068, Y1148, and Y1173) and minor (Y992) autophosphorylation sites of EGFR. These peptides may be exogenous substrates or pseudosubstrates that interfere with the autophosphorylation of EGFR. The effects of the peptides on autophosphorylation of EGFR were studied. 2. Purified EGFR was phosphorylated in vitro with EGF in the presence of various synthetic peptides. The phosphorylation level of EGFR was then evaluated after SDS-PAGE separation, followed by Western blot analysis with antiphosphotyrosine antibody. 3. Ac-VPEYINQ-NH2 (Y1068) and Ac-DYQQD-NH2 (Y1148) showed the most potent inhibitory effects, followed by Ac-ENAEYLR-NH2 (Y1173). These peptides at 4 mM suppressed phosphorylation to 30-50%. 4. Combination of the three kinds of peptides much more strongly inhibited autophosphorylation. The 50% inhibitory concentration (IC50) value was 0.5 mM as a mixture and was comparable to that of AG1478 (IC50, 0.3 mM) at 0.2 mM ATP. 5. Neither Ac-DIYET-NH2 or Ac-KIYEK-NH2, designed previously based on the amino-acid sequence of an autophosphorylation site of insulin receptor, nor their related (Ac-KIFMK-NH2) or unrelated (Ac-LPFFD-NH2) peptides showed an inhibitory effect. These results suggest that the small peptides that originated from the autophosphorylation sites of EGFR interact solely with EGFR. 6. The peptides containing the sequences surrounding Y1068, Y1148, and Y1173 may be a promising seed for the development of therapeutic agents for breast and lung cancers.