To investigate the regulatory mechanism of cell adhesion, we have searched for cellular inhibitory factors which prevent cell adhesion. The brain cytosol was found to inhibit the adhesion of various transformed cells to the substratum. An inhibitory 120-kDa protein was purified by sequential column chromatography. Peptide sequencing revealed that the protein is identical to amphiphysin 1. GST-amphiphysin 1 suppressed the attachment of HeLa cells to the plate when cells were cultured in the serum-containing medium. Vitronectin, a major cell-adhesive protein in serum and a ligand to alphanubeta3 integrin, was responsible for this cell attachment, and the vitronectin action was blocked by GST-amphiphysin 1. GST-amphiphysin 1 also inhibited the vitronectin-mediated spreading and migration of malignant melanoma cells. Furthermore, GST-amphiphysin 1 bound directly to vitronectin. These findings point to the interesting possibility that amphiphysin 1 could be a useful tool to inhibit cell-adhesive vitronectin. (C) 2003 Elsevier Science (USA). All rights reserved.