SummaryIn order to assess specific functional roles of plant heat shock transcription factors (HSF) we conducted a transcriptome analysis of Arabidopsis thaliana hsfA1a/hsfA1b double knock out mutants and wild‐type plants. We used Affymetrix ATH1 microarrays (representing more than 24 000 genes) and conducted hybridizations for heat‐treated or non‐heat‐treated leaf material of the respective lines. Heat stress had a severe impact on the transcriptome of mutant and wild‐type plants. Approximately 11% of all monitored genes of the wild type showed a significant effect upon heat stress treatment. The difference in heat stress‐induced gene expression between mutant and wild type revealed a number of HsfA1a/1b‐regulated genes. Besides several heat shock protein and other stress‐related genes, we found HSFA‐1a/1b‐regulated genes for other functions including protein biosynthesis and processing, signalling, metabolism and transport. By screening the profiling data for genes in biochemical pathways in which known HSF targets were involved, we discovered that at each step in the pathway leading to osmolytes, the expression of genes is regulated by heat stress and in several cases by HSF. Our results document that in the immediate early phase of the heat shock response HSF‐dependent gene expression is not limited to known stress genes, which are involved in protection from proteotoxic effects. HsfA1a and HsfA1b‐regulated gene expression also affects other pathways and mechanisms dealing with a broader range of physiological adaptations to stress.