NKX3.1 is a homeobox gene, expression of which is largely restricted to the adult prostatic epithelium. Loss of NKX3.1 expression has been linked to prostate carcinogenesis and disease progression and occurs in the absence of mutations in the coding region of the NKX3.1 gene. In this study, we have characterized regulation of NKX3.1 expression by all-trans retinoic acid (tRA), a naturally occurring vitamin A metabolite that is accumulated at high levels in the prostate. Using the prostate cancer cell line LNCaP, Western blot analysis revealed a approximately twofold induction of NKX3.1 protein levels following tRA exposure, with sequential analysis of NKX3.1 protein levels in cycloheximide co-treated cells indicating that tRA does not alter NKX3.1 protein turnover. The approximately 1.6-fold increase in NKX3.1 mRNA levels detected in tRA-treated LNCaP cells also occurred independently of new protein synthesis and was not mediated by changes in NKX3.1 mRNA stability. In contrast, nuclear run-on assays indicated that tRA treatment increased NKX3.1 transcription. To identify retinoid responsive regions of the NKX3.1 gene, DNA sequences encompassing approximately 2 kb of the NKX3.1 promoter or the entire 3'untranslated region (UTR) were cloned into luciferase reporter plasmids. Analysis of induced luciferase activity following transfection of these constructs into prostate cancer cells did not identify tRA responsiveness, however the 3'UTR was found to be strongly androgen responsive. These studies demonstrate that the NKX3.1 gene is a direct target of retinoid receptors and suggest that androgen regulation of NKX3.1 expression is mediated in part by the 3'UTR.