Chicken heart mesenchymal cells do not proliferate in medium of physiological composition containing plasma (S. Balk, Proc. Natl. Acad. Sci. USA 77:6606-6610, 1980). To understand the molecular events involved in cell quiescence and in the initiation of cell division under physiological conditions, we examined the differences in the patterns of protein synthesis of quiescent, hormone-stimulated, and Rous sarcoma virus-transformed chicken heart mesenchymal cells. We describe the expression of a 20,000-kilodalton (kDa) polypeptide actively synthesized by quiescent cells but not by their transformed counterparts. Normal chicken heart mesenchymal cells stimulated with epidermal growth factor and insulin also repressed the synthesis of the 20,000-kDa polypeptide while actively growing but synthesized increasing amounts of the protein at high cell density (confluence). The synthesis of the 20,000-kDa protein is not restricted to chicken heart mesenchymal cells, since confluent, density-arrested chicken embryo fibroblasts also expressed high levels of the protein. Transformed chicken heart mesenchymal cells and embryo fibroblasts did not synthesize the protein even at high cell density. The 20,000-kDa polypeptide accumulated in the culture medium.