The activated B-cell factor (ABF)-1 cDNA was initially isolated from Epstein-Barr virus (EBV)-infected B cells and codes for a DNA-binding protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors. In this study, we characterized the nuclear localization signal of ABF-1, mapped two distinct transcriptional repression domains, and identified one ABF-1-interacting protein, Id-2. By examining the subcellular location of deletion mutants of ABF-1 fused to green fluorescent protein (GFP), critical regions involved in nuclear localization were determined. Analysis of GFP-tagged ABF-1 deletion mutants revealed two separate regions capable of directing nuclear localization. One region mapped to the N-terminal amino acids 71 to 103, whereas the second region localized to the C-terminal bHLH domain. Transient transfection of ABF-1 deletion mutants demonstrated that the N-terminal amino acids 1 to 40 and the bHLH domain function together to achieve maximum repression of E2A activity. Taken together, these results indicate that ABF-1 is a nuclear transcriptional repressor with two distinct regions that function in a synergistic fashion to attenuate E2A-mediated gene activation.