The phospholipid composition of cell-substratum adhesion sites, obtained after EGTA-mediated detachment of cells from the tissue-culture substratum, was determined for [32P]orthophosphate radiolabeled Balb/c 3T3, SV40-transformed (SVT2), and concanavalin A selected revertant variant cell lines. All of the major phospholipid classes were found in the substrate-attached material, but there was an enrichment for specific phospholipid species in this adhesive material as compared to whole-cell and surface-enriched membranes. The phospholipid composition was remarkable similar for the whole-cell and surface-enriched membrane fractions from the three cell lines. However, pronounced differences in the phospholipid composition of the adhesion sites were observed as a result of viral transformation--SVT2 sites were clearly enriched in phosphatidylethanolamine and depleted in phosphatidylcholine when compared to 3T3 sites. This alteration in adhesion site phospholipids of transformed cells reverted to 3T3-like values in the adhesive material of revertant cells. The composition of adhesive material of newly attaching cells was also examined to differentiate compositional differences between "footpad" adhesion sites and "footprints", adhesive material pinched off from the posterior of cells as they move across the substratum. Pulse and pulse-chase analyses of the [32P]phospholipids revealed some differences in synthesis and turnover rates in the three cell lines; in addition, altered rates of deposition of newly synthesized material into adhesion sites of transformed cells were observed. These data afford further evidence that the cell-substratum adhesion sites are highly specialized areas of the cell surface enriched in components which are intricately involved in the adhesive process. The transformation-dependent changes in adhesion site phospholipids may help to determine the basis for the altered adhesive properties of transformed cells.