The estrogen response element (ERE) directly linked to a TATA box induces CAT activity in a hormone-dependent manner in Fe 33 cells, the rat hepatoma cell line FTO-2B, stably transfected with the human estrogen receptor (ER). The same promoter construct mediates the stimulation of in vitro transcription. This stimulation is dependent on the presence of the ERE. Induction of transcription in a variety of nuclear extracts derived from mammalian cells is of the same magnitude irrespective of the presence of ER. Similarly, transcription in vitro mediated by B1 vitellogenin 5' flanking sequences in different nuclear extracts is not due to the interaction of the ER with the ERE. Competition analyses with a variety of oligonucleotides reveal that proteins different from the ER, which recognize ERE-like DNA elements, functionally interact with the ERE in vitro. These experiments suggest that ubiquitous proteins related or even identical to the transcription factor USF (MLTF) activate in vitro transcription in an ERE-dependent manner.