In order to assess the specific functional roles of different plant heat shock transcription factors (HSFs) we have isolated T-DNA insertion mutants in the AtHsf1 and AtHsf3 genes of Arabidopsis thaliana. Complete and selective loss of the promoter binding activities of AtHSF1 or AtHSF3, verified by immunoprecipitation assays, had no obvious effects on the heat shock (HS) response in the individual mutant lines. Only hsf1(-) /hsf3(-)double mutants were significantly impaired in HS gene expression. In these plants the inability to form high-molecular-weight HSE-binding complexes correlates with a dramatic change in the kinetics of mRNA accumulation from all HSF target genes tested, including members of the Hsp100, Hsp90, Hsp70 and small Hsp families, and genes for two heat-inducible class B-HSFs. After prolonged HS, the amounts of most heat shock mRNAs expressed, except transcripts of Hsp18.2, reached approximately the same levels as in wild type plants. Our data indicate that AtHSF1 and AtHSF3 are key regulators of the immediate stress-induced activation of HS gene transcription, and consequently determine the kinetics of the negative feed back loop that is responsible for the transience of HS gene expression in wild type.