Stress agents known to elicit the unfolded protein response in Arabidopsis thaliana upregulate the expression of a constellation of genes dependent on the membrane-associated basic domain/leucine zipper (bZIP) transcription factor, bZIP28. Among the stress-activated genes, a consensus promoter sequence corresponding to the endoplasmic reticulum (ER) stress-responsive element I (ERSE-I), CCAAT-N10-CACG, was identified. Disruption of either the CCAAT or CACG subelement in ERSE-I resulted in reduction of the transcriptional response to ER stress. bZIP28 forms homo- and heterodimers with other bZIP TF family members (in subgroup D) and interacts with CCAAT box binding factors, heterotrimeric factors composed of NF-Y subunits. Arabidopsis encodes 36 NF-Y subunits, and it was found that subunits NF-YB3 and -YC2 interact with bZIP28 and NF-YA4, respectively, in a yeast three-hybrid system. A transcriptional complex containing bZIP28 and the above-mentioned three NF-Y subunits was assembled in vitro on DNA containing ERSE-I. bZIP28, on its own, binds to the CACG subelement in ERSE-I to form a smaller complex I, and in combination with the NF-Y subunits above, bZIP28 assembles into a larger transcriptional complex (complex II). bZIP28 was shown to interact with NF-Y subunits in vivo in bimolecular fluorescence complementation analyses and in coimmunoprecipitation assays. Treatment of seedlings with ER stress agents led to the upregulation of NF-YC2 and the relocation of NF-YB3 from the cytoplasm to the nucleus. Thus, in response to ER stress, bZIP28 is mobilized by proteolysis and recruits NF-Y subunits to form a transcriptional complex that upregulates the expression of ER stress-induced genes.