Ethylene-responsive factors (ERFs) are important regulators of plant gene expression.In this study,three novel ERF genes,GhERF2,GhERF3 and GhERF6,were isolated from cotton (Gossypium hirstum) using rapid amplification of cDNA ends-polymerase chain reaction.Transient expression analysis using GhERF-green fluorescent protein fusions showed that these three proteins were targeted to the nucleus.Fusion proteins consisting of GhERF2,GhERF3 or GhERF6 coupled to the GAL4 DNA binding domain strongly activated transcription in yeast.Furthermore,GhERF6 was shown to be able to bind specifically to GCC boxes using a particle bombardment assay in tobacco cells.Semi-quantitative reverse transcription-polymerase chain reaction revealed that GhERF2 and GhERF3 are constitutively expressed in all organs,while GhERF6 is only constitutively expressed in vegetative organs.When plants were treated with ethylene,abscisic acid,salt,cold and drought,the transcripts of GhERF2,GhERF3 and GhERF6 were rapidly induced to high levels.Promoter analysis also indicated that the 5 upstream regions of the three genes possess elements induced by these physiological and environmental factors.Collectively,our data suggest that GhERF2,GhERF3 and GhERF6 might function as positive trans-acting factors in the plant responses to ethylene,abscisic acid and other stresses and provide useful clues for further research into the mechanism of them in regulating cotton multiple stress responses. Ethylene-responsive factors (ERFs) are important regulators of plant gene expression.In this study, three novel ERF genes, GhERF2, GhERF3 and GhERF6, were isolated from cotton (Gossypium hirstum) using rapid amplification of cDNA ends-polymerase chain reaction. Transient expression analysis using GhERF-green fluorescent protein fusions showed those three proteins were targeted to the nucleus. Fusion proteins consisting of GhERF2, GhERF3 or GhERF6 coupled to the GAL4 DNA binding domain strongly activated transcription in yeast. Future Thermo, GhERF6 was shown to be able to bind specifically to GCC boxes using a particle bombardment assay in tobacco cells. Semi-quantitative reverse transcription-polymerase chain reaction revealed that GhERF2 and GhERF3 are constitutively expressed in all organs, while GhERF6 is only constitutively expressed in vegetative organs. with ethylene, abscisic acid, salt, cold and drought, the transcripts of GhERF2, GhERF3 and GhERF6 were rapidly indu ced to high levels. Promot analysis also indicated that the 5 upstream regions of the three genes possess elements induced by these physiological and environmental factors. Collectively, our data suggest that GhERF2, GhERF3 and GhERF6 might function as positive trans-acting factors in the plant responses to ethylene, abscisic acid and other stresses and provide useful clues for further research into the mechanism of them in regulating cotton multiple stress responses.