Ethylene-responsive factors (ERFs) have conserved DNA-binding domains and interact directly with the GCC box in the ethylene-responsive element that is necessary and sufficient for the regulation of transcription by ethylene. ERFs were shown to be localized to nucleus in transient transfection experiments. Transient expression assays using tobacco protoplasts and a heterologous system in yeast were used to examine the transactivation functions of ERFs. ERF2 and ERF4 enhanced the GCC box-mediated transcription of a reporter gene in tobacco protoplasts. When fused to the DNA-binding domain of yeast GAL4, a carboxy-terminal region of ERF2, as well as both amino-terminal and carboxy-terminal regions of ERF4, functioned as a transactivation domain in tobacco protoplasts. The amino-terminal regions of ERF2 and ERF4 functioned as transactivation domains in yeast. In contrast to ERF2 and ERF4, ERF3 reduced the transcription of the reporter gene in tobacco protoplasts, indicating that ERF3 functions as a repressor. Thus, it appears that ERFs exert their regulatory functions in different ways, with ERF2 and ERF4 being activators and ERF3 being a repressor of transcription.