NKX3.1 is a member of the NK class of homeodomain proteins and is most closely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exerts a growth-suppressive effect on prostatic epithelial cells and controls differentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequence that has not been reported for any other NK class homeoprotein. By electromobility shift assay we demonstrated that NKX3.1 preferentially binds the TAAGTA sequence rather than the binding site for Nkx2.1 (CAAGTG) or Msx1 (TAATTG). Using mutated binding sites in competitive gel shift assays, we analyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring polymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 was identical to the wild-type binding sequence. The binding affinities of wild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very similar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter vector with three copies of the NKX3.1-binding site upstream from a thymidine kinase promoter. The data show that among NK family proteins NKX3.1 binds a novel DNA sequence and can behave as an in vitro transcriptional repressor.