A variety of normal cells and tumor cell lines of differing metastatic potential were evaluated for their effect on various substrates consisting of purified preparations of the basement membrane-associated proteins fibronectin, laminin, and type IV collagen, which were labeled with tritium and/or rhodamine isothiocyanate. Different degrees of clearing or release of material from the substrate were observed, depending on the cell-protein combination. Normal fibroblasts as well as transformed cells and cells of low metastatic potential showed extensive clearing of surfaces coated with fibronectin, laminin, and type IV collagen. This clearing of protein began at sites of initial cell adhesion and was restricted to areas beneath and/or along the apparent paths of cell migration and beneath cellular processes. Covalent attachment of adhesion proteins to glass coverslips nearly eliminated clearing and release. Studies showed that a substantial amount of the fibronectin released from the substrate by HT-1080 fibrosarcoma cells is due to proteolysis. Release and clearing of substratum-attached protein is reduced by approximately 50% by antibodies specific for the protein on the substrate. Tumor cells with low metastatic potential were found to produce higher levels of clearing and release of protein adsorbed to the substrate than tumor cells with high metastatic potential. This was true for variants of the murine K-1735 melanoma and the UV-2237 fibrosarcoma with high and low metastatic capability on all three basement membrane-associated protein substrates. The differences in clearing and release between high and low metastatic cells were not due to differences in initial cell adhesion to the substrates but may be associated with differences in the affinity, type of cell-substrate interactions, proteases, or other variables.