SummaryUpon heat stress, heat shock factors (HSFs) control the expression of heat shock protein (HSP) genes by transcriptional activation. The perplexing multiplicity of HSF genes in Arabidopsis– 21 potential genes have been identified – renders it difficult to identify mutant phenotypes. In this study, we have attempted to generate a transdominant‐negative mutant of HSF by transgenic expression of a protein fusion construct, EN‐HSF1, consisting of the Drosophila engrailed repressor domain (EN) and the complete Arabidopsis AtHSF1. Transgenic lines were screened for impaired ability to induce high levels of low‐molecular‐weight heat shock proteins (sHSPs). Two lines, EH14‐6 and EH16‐3, which showed quantitative differences in the expression of EN‐HSF1, were further analysed for induction of thermotolerance and heat‐stress‐dependent mRNAs of a number of different HSF target genes encoding different HSP and HSF. The mRNA levels of all genes tested were moderately downregulated in EH14‐6 but strongly reduced in EH16‐3 plants compared to wild‐type (Wt) and HSF1‐overexpressing control plants. The inhibition of the induction of heat shock response correlated with impaired basal and acquired thermotolerance of the EH16‐3 line. The kinetics of HSP expression suggest that the negative effect of EN‐HSF1 is stronger in the early phase of the heat shock response, and that the reduction in mRNA levels is partially compensated at the translational level.