We have shown that the human general transcriptional factor IB (TFIIB) auto-acetylates specifically at lysine 238 in the presence of acetyl coenzyme A in vitro. This is the first case of acetylation of a transcription factor in the absence of a factor acetyltransferase (FAT). Acetylation of TFIIB results in a stronger interaction with transcription factor IIF (TFIIF) and activated transcription in vitro. Cells transfected with mutant TFIIB incapable of auto-acetylation show decreased levels of transcription in vitro. If auto-acetylation of TFIIB occurs in cells, acetyl coenzyme A levels may play an important role in the regulation of transcription. In addition, we report for the first time that the RAP30 subunit of TFIIF is also auto-acetylated in the presence of acetyl coenzyme A in a pH-dependent manner, similarly to TFIIB. This finding strongly suggests that auto-acetylation may be more important in regulating gene expression than previously believed.