The class A heat shock factors HsfA1a and HsfA1b are highly conserved, interacting regulators, responsible for the immediate-early transcription of a subset of heat shock genes in Arabidopsis. In order to determine functional cooperation between them, we used a reporter assay based on transient over-expression in Arabidopsis protoplasts. Reporter plasmids containing promoters of Hsf target genes fused with the GFP coding region were co-transformed with Hsf effector plasmids. The GFP reporter gene activity was quantified using flow cytometry. Three of the tested target gene promoters (Hsp25.3, Hsp18.1-CI, Hsp26.5) resulted in a strong reporter gene activity, with HsfA1a or HsfA1b alone, and significantly enhanced GFP fluorescence when both effectors were co-transformed. A second set of heat shock promoters (HsfA2, Hsp17.6CII, Hsp17.6C-CI) was activated to much lower levels. These data suggest that HsfA1a/1b cooperate synergistically at a number of target gene promoters. These targets are also regulated via the late HsfA2, which is the most strongly heat-induced class A-Hsf in Arabidopsis. HsfA2 has also the capacity to interact with HsfA1a and HsfA1b as determined by bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts and yeast-two-hybrid assay. However, there was no synergistic effect on Hsp18.1-CI promoter-GFP reporter gene expression when HsfA2 was co-expressed with either HsfA1a or HsfA1b. These data provide evidence that interaction between early and late HSF is possible, but only interaction between the early Hsfs results in a synergistic enhancement of expression of certain target genes. The interaction of HsfA1a/A1b with the major-late HsfA2 may possibly support recruitment of HsfA2 and replacement of HsfA1a/A1b at the same target gene promoters.