USF is a family of transcription factors that are structurally related to the Myc oncoproteins and also share with Myc a common DNA-binding specificity. USF overexpression can prevent c-Myc-dependent cellular transformation and also inhibit the proliferation of certain transformed cells. These antiproliferative activities suggest that USF inactivation could be implicated in carcinogenesis. To explore this possibility, we compared the activities of the ubiquitous USF1 and USF2 proteins in several cell lines derived from either normal breast epithelium or breast tumors. The DNA-binding activities of USF1 and USF2 were present at similar levels in all cell lines. In the non-tumorigenic MCF-10A cells, USF in general, and USF2 in particular, exhibited strong transcriptional activities. In contrast, USF1 and USF2 were completely inactive in three out of six transformed breast cell lines investigated, while the other three transformed cell lines exhibited loss of USF2 activity. Analyses in cells cultured from healthy tissue confirmed the transcriptional activity of USF in normal human mammary epithelial cells. These results demonstrate that a partial or complete loss of USF function is a common event in breast cancer cell lines, perhaps because, like Myc overexpression, it favors rapid proliferation.