The steady-state level and synthesis of a pair of polypeptides of Mr 33,000 and 35,000 in chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) are significantly decreased relative to normal CEF; however, the decrease is more pronounced in the case of the Mr 35,000 polypeptide. These polypeptides have been identified as the alpha and beta subunits of CEF tropomyosin by selective staining with tropomyosin antibody, two-dimensional gel electrophoresis, partial peptide analysis, and solubility properties. The decrease in tropomyosin is shown to be a transformation-specific phenomenon in that it does not occur after infection with a virus deleted in src sequences. Decreased synthesis of tropomyosin is also observed in quail cells transformed by MC29 (a retrovirus with a different onc gene than that in RSV) and also in chemically transformed quail cells. The decreased in tropomyosin is probably not a direct result of the disruption of the microfilament system in transformed cells because disruption of the microfilament system with trypsin or cytochalasin B in normal CEF does not lead to a decrease in tropomyosin synthesis. A decrease in tropomyosin in CEF after transformation may be a result of a pleiotropic effect that results in the transcriptional inactivation not only of the tropomyosin gene but also of the fibronectin and procollagen genes described by others.