Using two well-characterized heat stress transcription factors (Hsfs) from tomato (Lycopersicon peruvianum; LpHsfA1 and LpHsfA2), we analyzed the transcriptional activation of the Ha hsp17.6 G1 promoter in sunflower (Helianthus annuus) embryos. In this system, we observed transient promoter activation only with LpHsfA2. In contrast, both factors were able to activate mutant versions of the promoter with improved consensus Hsf-binding sites. Exclusive activation by LpHsfA2 was also observed in yeast (Saccharomyces cerevisiae) without other Hsfs and with a minimal Cyc1 promoter fused to the Ha hsp17.6 G1 heat stress cis-element. Furthermore, the same promoter mutations reproduced the loss of activation selectivity, as observed in sunflower embryos. The results of in vitro binding experiments rule out differential DNA binding of the two factors as the explanation for the observed differential activation capacity. We conclude that the specific sequence of this heat stress cis-element is crucial for Hsf promoter selectivity, and that this selectivity could involve preferential transcriptional activation following DNA binding. In sunflower embryos, we also observed synergistic transcriptional activation by co-expression of LpHsfA1 and LpHsfA2. Mutational analyses of the Ha hsp17.6 G1 promoter, combined with in vitro binding assays, suggest that mixed oligomers of the two factors may be involved in promoter activation. We discuss the relevance of our observations for mechanisms of developmental regulation of plant heat stress protein genes.