A new member of the mouse NK family of homeobox genes that is related to Drosophila NK-3 has been identified. Expression of this gene, termed Nkx-3.1, is largely restricted to the prostate gland in adult animals. The level of Nkx-3.1 mRNA decreases markedly in response to castration, suggesting that its expression is androgen-dependent. In situ hybridization analyses demonstrated that expression of Nkx-3.1 in the prostate is confined to epithelial cells. In newborns, Nkx-3.1 mRNA is detected in the urethral epithelium that is being induced by the surrounding mesenchyme to invaginate to form prostatic buds. Together, these observations suggest that the Nkx-3.1 protein, which likely functions as a transcription factor, plays a prominent role both in the initiation of prostate development and in the maintenance of the differentiated state of prostatic epithelial cells.