RNA helicase A (RHA) is a member of ATPase/helicase and regulates the transcription through recruitment of Pol II and/or by ATP dependent mechanisms. In CREB-dependent transcription, RHA recruits RNA polymerase (Pol) II to the CREB binding protein (CBP) via the minimal transactivation domain (MTAD). This region is well conserved among RHA homologues, whereas it is unique to RHA. The three conserved tryptophan residues in MTAD are critical for transactivation. To understand the importance of tryptophan residues on transactivation, we generated mutants in which tryptophan residues were replaced by other aromatic, bulky hydrophobic or small hydrophobic amino acids. Substitutions of tryptophan with either bulky hydrophobic or small hydrophobic amino acid decreased transcriptional activity, whereas aromatic residue had no effect. Moreover, these mutants with tryptophan to phenylalanine, activated CREB-dependent transcription. These results indicate that aromatic characteristics of tryptophan residues in MTAD are important for CREB-dependent transcription via RHA.