We have found that cytoskeletal extracts of cultured chicken embryo fibroblasts contain at least seven distinct polypeptides (two major and five minor) which cross-react with antiserum to chicken smooth muscle tropomyosin. These polypeptides range in apparent molecular weight from 31,000 to 47,000, and each is encoded by mRNAs which specifically hybridize to cloned muscle tropomyosin cDNAs. These nonmuscle tropomyosin species and their respective mRNAs are electrophoretically distinct from those of chicken skeletal muscle and appear by genomic DNA blotting to comprise a part of a multigene tropomyosin family. In Rous sarcoma virus-transformed chicken embryo fibroblasts, synthesis of the tropomyosins is differentially repressed such that the synthesis of the major species (cp35 and cp33, cytoskeletal proteins of molecular weight 35,000 and 33,000, respectively) and three minor species is drastically reduced, whereas the synthesis of two of the minor species (cp32 and cp31) remains essentially unchanged. Analysis of cellular mRNA and runoff nuclear transcription experiments indicate that the repression of tropomyosin synthesis by Rous sarcoma virus transformation occurs at the level of transcription. This repression of tropomyosin synthesis is partially mimicked in normal chicken embryo fibroblasts during incubation in high-NaCl medium, a condition in which chicken embryo fibroblasts acquire many characteristics of transformed cells.