Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::beta-glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.