Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein. Deletion mutagenesis revealed amino acids 2484-2520 in human ATBF1 and 279-300 in v-Myb as regions required for in vitro binding of both proteins. Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity. Both ATBF1 2484-2520 and Myb 279-300 regions were required for the inhibitory effect. Finally, the chicken ATBF1 was identified, showing high degree of amino acid sequence homology with human and murine proteins. Our data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279-300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated.