Activator-dependent transcription in mammalian cells requires upstream stimulatory activity (USA)-derived cofactors in addition to those present in TFIID. A novel positive cofactor (PC4) purified from the human USA fraction effected a marked enhancement (up to 85-fold) of GAL4-AH-dependent transcription in conjunction with TFIID and other general factors. Isolation of a corresponding cDNA identified PC4 as a 127 residue single-stranded DNA-binding protein with serine-rich regions near the N-terminus. Recombinant PC4 was functionally equivalent to native PC4, and both proteins markedly enhanced activation by diverse activation domains fused to the DNA-binding domain of GAL4. Recombinant PC4 interacted independently both with free or DNA-bound VP16 activation domains and with free or DNA-bound TFIIA-TBP complexes (but not with TBP alone). These results indicate that PC4 is a general coactivator that functions cooperatively with TAFs and mediates functional interactions between upstream activators and the general transcriptional machinery.