Transcription of a proto-oncogene c-fos is induced rapidly to high levels by various extracellular stimuli. To explore the molecular mechanism of c-fos gene induction, we established a defined in vitro transcription system for the c-fos promoter that consists of purified activators (SRF, Elk-1, cAMP-responsive element-binding protein, and ATF1), general transcription factors, and RNA polymerase II. In this reconstituted transcription system, activation of c-fos transcription was highly dependent upon coactivators such as PC4 and Mediator, indicating a very weak activation potential of the activators in the context of an unaltered promoter structure. This heightened coactivator dependence, however, allowed us to identify from HeLa nuclear extract a coactivator-like activity termed transcriptional regulator of c-fos (TREF) that enhanced c-fos transcription but not GAL4-VP16-dependent transcription. TREF cooperated with Mediator to enhance c-fos transcription by approximately 60-fold over its basal level and, like Mediator, stimulated activator-independent (basal) transcription as well. Further purification of TREF revealed that it consists of at least three distinct components, one of which was purified to near homogeneity and identified as heterogeneous nuclear ribonucleoprotein R. Recombinant heterogeneous nuclear ribonucleoprotein R enhanced transcription from the c-fos promoter and displayed cooperativity with PC4 and Mediator, thus demonstrating its direct transcriptional activity.