Integrins are widely expressed cell surface molecules that mediate cell attachment to extracellular matrix (ECM) proteins. They also interact with molecules on their own membranes, and these cis-interactions play a crucial role in integrin-dependent cellular responses. We herein analysed what molecules interact with β1 integrin during biological events induced by cell attachment to different ECM proteins, using a recently established reaction, the enzyme-mediated activation of radical sources (EMARS). The interactions between β1 integrin and receptor tyrosine kinases including EGFR and ErbB4 reached a peak at 2 h after seeding HeLa S3 cells onto the ECM proteins. The peak of phosphorylation of ErbB4 (at 2 h after seeding the cells onto fibronectin) coincided with the peak of the interaction with β1 integrin, while that of EGFR (at 1 day) did not. Accompanying with these findings, suppression of cell migration by a pharmacological inhibitor of the ErbB family receptors, PD168393 and an anti-ErbB4 neutralizing antibody, 12D8 was observed at 2 h after seeding. Taken together, it is deduced that interactions between β1 integrin and ErbB4 occur in a spatiotemporally-regulated manner, and such interaction contributes to the integrin-dependent cell migration.