Upon germination of seed cereals, mobilization of the reserves stored in the endosperm is regulated by the phytohormones gibberellins (GA) and abscisic acid (ABA). In barley, the cis regulatory elements and the trans-acting factors mediating the ABA response of hydrolase genes remain elusive. Two new barley genes, HvDof17 and HvDof19, encoding transcription factors (TFs) of the DNA binding with one finger (DOF) class have been characterized and their role upon germination investigated. HvDOF19 binds in a specific manner to the pyrimidine box within the GARC of a thiol-protease gene (Al21), and mediates the ABA repression of this gene in the barley aleurone. Silencing of HvDof19 in transient expression assays diminishes the inhibitory effect of ABA upon expression of the Al21 gene promoter. Transcripts from HvDof17 and HvDof19 accumulate early in germinating aleurones with a peak at 16 h after seed imbibition (hai), whereas the mRNAs of the GA-induced activator GAMYB remain little expressed. At 48 hai, mRNA content of both genes is comparatively insignificant compared with that of GAMYB, which reaches a maximum. Both TFs repress, in transient expression assays, the GA- and the GAMYB-mediated activation of this thiol-protease gene (Al21). In addition, HvDOF17 and HvDOF19 interact with GAMYB in plant cell nuclei, and HvDOF17, but not HvDOF19, interferes with the DNA binding of GAMYB to its target site in the promoter of the Al21 gene. A regulatory model of hydrolase gene expression upon germination is proposed.