Transient expression analyses in sunflower embryos demonstrated that ABI3, a seed-specific transcription factor from Arabidopsis, activated chimaeric genes with the Ha hsp17.7 G4 promoter. Nucleotide substitutions at crucial positions of heat shock cis-elements established that they are required for the transcriptional activation involving ABI3. Trans-activation with Lp-HSFA1, a heat shock factor from tomato, reproduced the activation patterns of wild-type and mutant promoters observed with ABI3. In addition, ABI3 and Lp-HSFA1 synergistically activated the Ha hsp17. 7 G4 promoter, but only when it contained the intact proximal and distal heat shock cis-elements. The activation domain of Lp-HSFA1 was necessary for promoter activation. An amino terminal deletion of ABI3 had dominant negative effects on activation by Lp-HSFA1. We failed to detect a substantial transcriptional activation by ABI3 in the absence of either functional heat shock factors or heat shock elements (HSEs). Furthermore, the wild-type, but not the mutant HSEs (from - 136 to - 49 in Ha hsp17.7 G4) were sufficient, in the context of a - 46 CaMV 35S promoter, to support activation by Lp-HSFA1, or Lp-HSFA1 and ABI3. These results demonstrate, for the first time, transcriptional activation of a heat shock protein promoter by ABI3. We also suggest that ABI3 functions as a transcriptional co-activator through heat shock factors.