NKX3A (NKX3.1) is a recently identified androgen-regulated gene that is largely specific to prostate for expression and likely to code for a homeobox protein. Here we report the full-length mRNA and genomic organization of human NKX3.1. There are at least five different splice variants of NKX3.1 mRNA that result in different open reading frames (ORFs). There is extensive similarity between the human and the mouse NKX3.1 cDNA sequences outside of the ORFs (greater than 60% overall identity), which may be involved in modulating NKX3.1 expression. In addition to its androgen regulation in the prostate cancer cell line LNCaP, we show that NKX3.1 expression is androgen-dependent in the CWR22 prostate cancer xenograft model. Interestingly, NKX3.1 is highly expressed in the androgen-independent derivative CWR22R in the absence of androgens, indicating that it may be deregulated in advanced prostate cancer. Using a Green Fluorescent Protein fusion construct, we show that NKX3.1 is a nuclear protein consistent with its proposed function as a homeobox transcription factor. Furthermore, in addition to androgens, NKX3.1 expression is up-regulated by 17beta-estradiol, but not by progesterone, dexamethasone, or 3,5,3'-triiodothyronine in LNCaP cells. Regulation of NKX3.1 by androgens and 17beta-estradiol in prostate cancer cells and its deregulation in androgen-independent prostate cancer suggest that it may have important regulatory roles during prostate cancer progression.