We previously isolated different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. We now report that NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, we determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia.