To characterize differences in gene expression between hormone-dependent and hormone-independent mammary carcinoma, we cloned complementary DNAs of genes expressed in a hormone-independent breast carcinoma cell line that were not expressed in a hormone-dependent line. One clone, which was isolated in many copies, coded for the intermediate filament protein vimentin. A complementary DNA clone 1.8 kilobases long included the entire protein-coding region for vimentin. Vimentin was expressed by more than one-half of the hormone-independent breast carcinoma cell lines tested but not by the hormone-dependent cell lines. The cell lines which expressed vimentin expressed only low levels of cytokeratins. The correlation between vimentin expression and more advanced stages of mammary cell transformation was tested in a model system in which immortal, nontumorigenic human mammary epithelial cells or derivative lines transformed with v-ras-H or SV40 T-antigen were found not to express vimentin, whereas a derivative highly tumorigenic cell line transformed by both v-ras-H and T-antigen did express vimentin. Analysis of several other kinds of epithelial carcinoma cell lines showed only rare examples of vimentin expression.