The interaction of cultured cells with their growth substrata has been studied as a function of oncogenic transformation by using chicken embryo fibroblasts infected with the temperature-sensitive mutant of Rous sarcoma virus, LA24, and grown in plastic culture dishes. In comparison to total cell fractions, substratum-associated material (SAM), prepared by EGTA release of transforming cells from culture dishes, is enriched in a 21-kilodalton (kDal) protein. Synthesis and deposition of this protein in SAM are stimulated within hours of transfer of cells to the permissive temperature (35°C), peak around 8 hr, and decline to levels 1.3-fold higher than those of controls at 41°C by 20 hr after the temperature shift. In contrast, incorporation of ³H-labeled amino acids into newly synthesized fibronectin in SAM is not significantly influenced by the transformation process during this time. Furthermore, although the presence of fibronectin in SAM is influenced by cell density, the 21-kDal protein is increased in SAM of transforming cells at all densities examined. The 21-kDal protein is not present in increased amounts in SAM from normal, uninfected chicken embryo fibroblasts grown at 41°C and 35°C or from cells infected with the wild-type Rous sarcoma virus (Prague A), which are fully transformed. It is not a mannose-containing glycoprotein and does not appear to be phosphorylated. Furthermore, it is not a product of normal cell protein degradation induced by transformation but results from de novo protein synthesis after shift of LA24-infected cells to the permissive temperature. Finally, turnover of the 21-kDal protein is slower at 35°C than at 41°C. This amplifies the effect of increased synthesis and results in a net accumulation in SAM during the early stages of transformation.