AbstractNKX‐3.1 is a suspected prostate tumor suppressor gene that encodes a homeodomain transcripti on factor. NKX‐3.1 has been demonstrated to interact with prostate derived Ets factor (PDEF) and to suppress the ability of PDEF to transactivate the prostate specific antigen promoter. To dissect the molecular basis of the interaction between these transcription factors, deletion analyses were preformed using the yeast two‐hybrid system. The interaction of NKX‐3.1 with full‐length PDEF requires part of the homeodomain and a tyrosine‐rich 21 amino acid sequence that lies C‐terminal to the homeodomain. The interaction of PDEF with full‐length NKX‐3.1 requires the Ets domain and a linker region that lies between the Ets and pointed domains. Deletion of the C‐terminal 21 amino acids of NKX‐3.1 completely disrupts the ability to suppress the transactivation function of PDEF in prostate tumor cells, demonstrating concordance between interaction in yeast and function in mammalian cells. These studies have identified novel protein–protein interaction domains within NKX‐3.1 and PDEF that operate in concert with their respective DNA binding domains to mediate functional interactions between these growth regulatory transcription factors. © 2004 Wiley‐Liss, Inc.