The four HD2 proteins of Arabidopsis thaliana (AtHD2A-D) belong to a unique class of histone deacetylases that is plant specific. Previously, we have demonstrated that one of the members, AtHD2A, can mediate transcriptional repression when targeted to the promoter of a reporter gene. Here, we report that AtHD2B and AtHD2C can also repress gene expression. AtHD2A and AtHD2C differ from AtHD2B and AtHD2D in the composition of their structural domains. Our data show that both structural types play a role in the repression of gene transcription. We demonstrate that AtHD2A can mediate gene repression through interactions with transcription factors in plants. By fusing AtHD2A with the DNA-binding domain of the plant transcriptional factor Pti4, the expression of a GCC box containing reporter gene was repressed. We also demonstrated repression of a GUS gene with GAL4 enhancers using transgenic plants that expressed a GAL4/AtHD2A fusion gene. Furthermore, the expression of the GAL4/AtHD2A protein using the seed-specific napin promoter (NAP2) and the constitutive tCUP promoter demonstrated that repression of transgenes could be achieved in a tissue-specific or unrestricted manner. Targeting of HD2 proteins to specific promoters using transcription factor DNA-binding domains may therefore provide a new technology for silencing target genes and pathways in plants as well as for assessing the function of unknown transcription factors.