Heat shock transcription factor A2 (HsfA2) is induced under environmental stress and regulates transcription of various defense-related genes. Thus HsfA2 plays an important role in induction of defenses against different types of environmental stress, but its mode of regulation remains unknown. To clarify the signal transduction pathway involved in the regulation of HsfA2 expression, we investigated the effect of MG132, a 26S proteasome inhibitor, or geldanamycin (GDA), a heat shock protein 90 (Hsp90) inhibitor, on the transcription of HsfA2 and its targets, Hsp18.1-CI and ascorbate peroxidase 2 (Apx2), in Arabidopsis T87 cells. The levels of transcripts were significantly increased by treatment with MG132 or GDA. Overexpression of a dexamethazone-inducible dominant-negative form of Hsp90.2 in Arabidopsis plants caused significant expression of HsfA2 and its target gene on treatment with the compound. Treatment with MG132 or GDA had no effect on intracellular levels of reactive oxygen species (ROS). Interestingly, the levels of polyubiquitinated proteins as well as the levels of HsfA2 transcript were rapidly increased under oxidative stress derived from treatment with H2O2 or methylviologen, while they were completely suppressed by pre-treatment with ascorbate, a scavenger of ROS, under oxidative stress. The present findings suggest that the inhibition of 26S proteasome function and/or Hsp90 activity is involved in the induction of HsfA2 expression in response to oxidative stress.