The genes and proteins of the HSP70 family, are involved in important processes in cells and organelles at normal temperature and after heat stress. Constitutive Hsc70 and heat-inducible Hsp70 genes are known in all organisms including plants. The goal of our present investigation was to generate an Hsp70 mutation in Arabidopsis thaliana. In a transgenic approach a heat-inducible antisense Hsp70 gene was constructed, plants were transformed and screened for lack of heat-inducible HSP70 mRNA; two such lines were further investigated. In these plants the Hsp70 gene was not induced by heat shock, and the level of HSC70 RNA was also greatly reduced. This negative antisense effect was specific for genes of the HSP70 family and the induction of mRNAs encoding the small HSP18 class of heat shock protein (HSP) was not affected. The level of HSP70/HSC70 proteins was significantly reduced in transgenic plants, but HSP18 was induced to the same level in different transgenic lines and in untransformed plants. The acquisition of thermotolerance was negatively affected in artisense plants, the survival temperature being 2 degrees C below the survival temperature of the wild type and other transgenic lines. Another major effect concerning the regulation of the endogenous heat shock transcription factor HSF was detected by testing the ability to form heterotrimers between authentic HSF and recombinant HSF-GUS (beta-glucuronidase) proteins. The shut-off time, required to turn of HSF activity during recovery from heat stress, was significantly prolonged in antisense plants compared with wild-type and other transgenic lines. Our results imply a dual role of HSP70 in plants, a protective role in thermotolerance and a regulatory effect on HSF activity and hence the autoregulation of the heat shock response.