The immediate-early 2 (IE2) protein of human herpesvirus 6 (HHV-6) is a potent transactivator of multiple cellular and viral promoters. Deletion mutants of HHV-6 variant A IE2 allowed us to map functional transactivation domains acting on complex and minimal promoter sequences. This mapping showed that both the N-terminal and C-terminal domains of IE2 are required for efficient transactivation, and that deletion of the C-terminal (1397-1466) tail of IE2 drastically reduces both transactivation and the intranuclear distribution of IE2. Moreover, we determined that the ATF/CRE binding site within the HHV-6A polymerase promoter is not required for efficient transactivation by IE2, whereas the R3 repeat region of the putative immediate-early promoter of HHV-6A is responsive to and positively regulated by IE2. These results contrast sharply to that of human cytomegalovirus (HCMV) IE2, which down-regulates its promoter. Our characterization of HHV-6 IE2 transactivating activity provides a better understanding of the complex interactions of this protein with the viral and cellular transcription machinery and highlights significant differences with the IE2 protein of HCMV.