The proteins which have been left tightly bound to the tissue culture substrate after ethylenebis (oxyethyl-enenitrilo) tetraacetic acid (EGTA)-mediated removal of normal, virus-transformed, and revertant mouse cells and which have been implicated in the substrate adhesion process have been analyzed by slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three size classes of hyaluronate proteoglycans were resolved in the 5% well gel; approximately half of the protein in the substrate-attached material coelectrophoresed with these polysaccharides-so-called glycosaminoglycan-associated protein(GAP). A portion of the GAP was shown to be highly heterogeneous and displaced from the polysaccharide by preincubation with calf histone before electrophoresis. The relative proportions of the proteoglycans varied in material deposited during a variety of cellular attachment and growth conditions. The remainder of the cellular protein in substrate-attached material was resolved as several major and distinct protein bands in 8 or 20% separating gels (a limited number of distinct serum proteins have also been identified as substrate bound). Protein C0 (molecular weight 220 000) was a prominent component in the material from a variety of normal and virus-transformed cells and resembled the so-called LETS or CSP glycoprotein in several respects; protein Ca was myosin-like in several respects; protein C2 was shown to be actin; and protein C1 (molecular weight 56 000) does not appear to be tubulin. Histones were also present in most preparations of substrate-attached material, particularly at high levels in transformed cell meterial, and may result from EGTA-mediated leakiness of the cell and subsequent binding to the negatively charged polysaccharide. These substrate-attached proteins were (a) prominent in substrate-attached material from many cell types in characteristic relative proportions, (b) deposited by EGTA-subcultured cells during the first hour of attachment to fresh substrate, (c) deposited by cells growing on plastic or glass substrates (three additional) components were also prominent in glass-attached material), and (d) deposited during long-term growth on or initial attachment to substrates coated wit 3T3 substrate-attached material. Pulse-chase analyses with radioactive leucine indicated that these proteins exhibit different turn-over behaviors. These results are discussed with regard to the possible involvement of these substrate-attached proteins in the substrate adhesion process, with particular interest in the interaction of cytoskeletal microfilaments with other surface membrane components and with regard to alteration of substrate adhesion by virus transformation.